KR101261000B1 - Composition for improving blastocyst hatching and trophectoderm differentiation comprising Dickkopf-1 and method thereof - Google Patents

Abstract

The present invention relates to a composition for improving blastocyst hatching rate and TE differentiation rate and a culture method thereof, and more particularly to a composition for improving blastocyst hatching rate and TE differentiation rate including Dickkopf-1 and a method for culturing the same. will be.

Description

Composition for improving blastocyst developmental ability comprising Dixivacox-1 and its method {composition for improving blastocyst hatching and trophectoderm differentiation comprising Dickkopf-1 and method}

The present invention relates to a composition for improving blastocyst hatching rate and TE differentiation rate and a cultivation method thereof, and more particularly, to improve blastocyst hatching rate and TE differentiation rate including Dickkopf-1 (hereinafter, referred to as 'Dkk1'). It relates to a composition for and a culture method thereof.

The birth of cloned sheep Dolly in 1997 (Wilmut I., Schnieke AE., McWhir J., Kind AJ., Campbell KHS.Viable offspring derived from fetal and adult mammalian cells.Nature 385: 810-13 (1997)) It is the achievement of advanced biotechnology that shows that when somatic cells meet with an egg, they are able to dedifferentiate to the embryonic stage and then divide into one complete individual to form a clone, ie omnipotent.

Stem cells are cell types that have the unique ability to regenerate themselves and give rise to evolved or differentiated cells. Most cells in the body, such as heart cells or skin cells, are dedicated to performing a particular function, but stem cells are not entwined until they are signaled to develop into differentiated cell types. What makes the stem cells unique is their proliferative capacity as well as the ability to differentiate. For years, researchers have focused on finding ways to use stem cells to replace damaged or dead cells and tissues. To date, most studies have focused on two types of stem cells: embryonic and adult stem cells. Embryonic stem cells are derived from preimplanted fertilized oocytes, ie blastocysts, while adult stem cells are present in mature organisms, for example in the bone marrow, epidermis and intestine. According to many national laws of Europe and other countries, fertilized oocytes are not recognized as embryos before implantation in the uterus, ie 10 to 14 days after fertilization, so that such cells are derived from stem cells or hBS derived from blastocysts. Called a cell. Pluripotency tests demonstrated that the embryo or blastocyst-derived stem cells can give rise to all cells in an organism, including embryonic cells, while adult stem cells have a more limited repertoire to the cell types to which they are delivered.

Somatic cloning technology refers to using somatic cells as donor nuclei, which are recovered from adult or fetal tissue. Nucleated oocytes to which the donor nucleus is to be transplanted must have the capacity to develop after fusion with somatic cells. In order to have this ability, the development of the egg must proceed until the second stage of meiosis (the time when the egg is naturally ovulated in the body). For this purpose, the egg may be artificially cultured outside the body or through hormonal treatment. Develop.

The recovered eggs undergo denuclearization prior to fusion with the somatic cells, and are usually removed using a physical method to remove the nucleus of the egg (the cytoplasm of the second meiosis intermediate substrate). After denuclearization, the oocytes are transplanted by somatic cell transplantation into the egg cavity between the zona pellucida and the oocyte cytoplasmic membrane or by direct intracellular injection.

In order to prevent chromosomal damage due to somatic cell nuclear transfer and to improve the developmental potential after transplantation, the cell cycle of somatic cells is adjusted to G0 group before somatic cell transplantation. However, this process can be omitted depending on the characteristics of the somatic cells to be transplanted, and in some cases, G2 and M cells have been reported to be normally generated.

After somatic cell donor nuclei are injected into denuclear eggs, they are physically fused through electrical stimulation, etc. Afterwards, fused cells with somatic cells are called somatic cell cloning embryos. After the production of somatic cell embryos, the most important process is that the transplanted somatic cell nucleus performs a series of developmental processes in harmony with the cytoplasm of the egg, with the same ability and function as the nucleus produced by sperm and oocyte nucleus conjugation. This series of processes is called reprogramming, and the process of inducing segmentation, which is the first occurrence of replication eggs, is called activation. Chemical or electrical stimulation is used for activation, and many studies have been conducted to induce normal reprogramming of cloned embryos after division. In the case of cloned fertilized eggs, which are reprogrammed smoothly, they are cultured in vitro until the blastocyst during differentiation (differentiation) for the first time.

A series of studies are currently underway to increase the efficiency of somatic cloning techniques. For example, studies on the expression of genes regulating early embryonic development, studies on methylation patterns used as indicators of reprogramming of fertilized eggs, morphological studies on microstructure abnormalities of organelles through electron microscopy, blastocysts. Optimal conditions for denuclearization, donor cell types and in vitro culture, fusion, activation, and in vitro culture of cloned eggs through experimental results derived from studies on the number and ratio of inner cell mass and feeder cells By setting up to increase the production efficiency of somatic stem cells derived from fertilized eggs.

In particular, in studies to improve embryonic development, inadequate reprogramming has been reported to be an important factor in reducing embryonic developmental efficiency (William MR., Kevin E., Rudolf J. Nuclear cloning and epigenetic). reprogramming of the genome.Science 293: 1093-1098 (2001); Kang YK., Koo DB., Park JS., Choi YH., Ahung AS., Lee KK., Han YM.Aberrant methylation of donor genome in cloned bovine embryos.Nature Genetics 28: 173-177 (2001). In addition, it has been reported that not only the methodological aspects of somatic cell nuclear transfer technology but also the extracellular environment such as in vitro culture conditions are important factors in reducing the developmental delay of cloned embryos (Shin SJ., Lee BC., Park JI., Lim JM., Hwang WS.Theriogonology 55: 1697-1704 (2001)

On the other hand, the incidence of apoptosis in embryonic blasts is a major factor in lowering early embryonic development of fertilized eggs (Handyside AH., Hunter S. Cell division and death in the mouse blastocyst before implantation. Rouxs Arch Dev Biol 195: 519-526 ( 1986); Jurisicova A., Varmuza S., Casper RF.Programmed cell death and human embryo fragmentation.Mol Hum Reprod 2: 101-106 (1996); Jurisicova A., Varmuza S., Casper RF.Involvement of programmed cell death in preimplantation embryo demise.Hum Reprod Update 1: 558-566 (1995)).

Therefore, even in the case of cell embryos, apoptosis in embryonic blasts is assumed to be the main cause of lowering the early developmental rate of embryos. Therefore, the focus should be on improving embryo development rate by inhibiting apoptosis in vitro. .

For example, according to previous studies, in vitro fertilization improved the development rate of in vitro fertilized eggs when incubated with nitric oxide scavenger or antioxidant, which is believed to inhibit apoptosis during in vitro culture (Takahashi). M., Keicho K., Takahashi H., Ogawa H., Schultz RM., Okano A. Theriogenology 54: 137-145 (2000); Lim and Hansel.Mol Reprod Dev 50: 45-53 (1998); Park SE , Chung HM., Ko JJ., Lee BC., Cha KY., Lim JM. Fertil Steril 74: 996-1000 (2000); Takahashi Y., First NL.Theriogenology 37: 963-978 (1992); de Matos DG., Furnus CC. Theriogenology 53: 761-771 (2000).

SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned problems and has been devised by the above necessity, and an object of the present invention is to provide a composition for improving blastocyst hatching rate.

Another object of the present invention is to provide a method for improving blastocyst hatching rate.

Another object of the present invention is to provide a composition for improving trophectoderm differentiation.

Another object of the present invention is to provide a method for enhancing trophectoderm differentiation.

In order to achieve the above object, the present invention provides a composition for improving blastocyst hatching rate comprising Dickkopf-1 as an active ingredient.

In one embodiment of the present invention, the blastocyst is preferably a mammalian blastocyst, more preferably a pig blastocyst, but is not limited thereto.

In one embodiment of the present invention, the Dickkopf-1 is preferably the polypeptide described in SEQ ID NO: 1, but all Dickkopf-1 or functional fragments and variants thereof exhibiting the desired effect of the present invention are also Dickkopf- of the present invention. It is included in 1 range.

In another aspect, the present invention is a method for culturing a mammal embryo, by adding Dickkopf-1 to the culture medium to increase the blastocyst hatching rate of the mammal, characterized in that to increase the rate of blastocyst hatching during development of unit embryos Provide a method.

In one embodiment of the method of the present invention, the Dickkopf-1 is preferably 24 hours to 48 hours, but is not limited thereto.

In another aspect, the present invention provides a composition for promoting trophectoderm differentiation comprising Dickkopf-1 as an active ingredient.

In another aspect, the present invention is a method for culturing mammalian embryos, by adding Dickkopf-1 to the culture medium to increase the rate of blastocysts hatched during the development of unit embryos, culturing for promoting trophectoderm differentiation of mammals Provide a method.

Hereinafter, the present invention will be described.

We investigated the potential role of the Wnt signaling pathway during early trophectoderm differentiation of preimplantation blastocysts in pigs. We found that Wnt signaling directly or indirectly affects blastocyst hatching.

First, the present inventors measured the effects of Dickkopf-1 (Dkk1) and lithium chloride (LiCl), inhibitors and activators of the Wnt signaling pathway, respectively, in the development of in vitro blastocysts. The results showed a significant increase in blastocyst hatching rate (83.6 ± 5.2 vs. 58.7 ± 4.8%) during the treatment of 10 ng / ul Dkk1 for 48 hours in the culture medium compared to the control, while significantly decreased during 10 mM LiCl feed. (17.4 ± 1.2 vs. 68.2 ± 2.0%).

In addition, to investigate the correlation between Wnt signaling and ectoderm differentiation (TE) differentiation in blastocyst development, we incubated the loss embryos and early blastocysts with Dkk1 for 24 hours, and the blastocyst hatching rate, total cell number per blastocyst It was confirmed that the ratio increased. The mRNA expression level of CDX2 gene, which plays a critical role for TE cell differentiation, was higher in Dkk1 treated blastocysts than in control or LiCl treated blastocysts. Overall, the inventors have found that down regulation of Wnt signaling promotes development of blastocysts and TE differentiation in pigs.

Hereinafter, the present invention will be described in detail.

We used Dickkopf-1 (Dkk1), an inhibitor that inhibits the accumulation of active β-catenin in both plasma and nuclei at different concentrations (0, 0.1, 1.0, and 10 ng / μl) in NCSU23 culture medium from day 6 to 24 hours. ). As can be seen from Table 2, blastocyst hatching rate increased dose-dependently. There was a significant difference in blastocyst hatching rates between the control group and the 10 ng / μl Dkk1 treated group (4.8 ± 4.2 vs. 26.2 ± 6.0%). In contrast to blastocyst hatching rate, blastocyst expansion rate was significantly decreased in the 10 ng / μl Dkk1 treatment group (57.1 ± 8.7 vs. 76.2 ± 6.8%) compared to the control treatment group.

Table 2 shows the effect of Dickkopf-1 (Dkk1) on the hatching capacity of pig blastocysts after 24 hours from day 6 at various concentrations in NCSU23 medium.

In order to confirm its effect on the incubation of blastocysts, we measured the blastocyst hatching rate between the control and 10 ng / μl Dkk1 treatment groups. On day 6 blastocysts were cultured continuously for 24 and 48 hours in the presence or absence of Dkk1. The results showed that the blastocyst hatching rate was significantly higher in the Dkk1 treated group than in the control group (58.7 ± 8.0 vs. 83.6 ± 5.5%; FIGS. 1A, b).

In particular, the level difference increased rapidly during 24 hours after Dkk1 treatment, but remained relatively constant between 24 hours and 48 hours (FIG. 1A). These results indicate that Dkk1 treatment promotes or induces early entry into the process of hatching blastocysts.

To determine the effect of LiCl on the hatching ability of in vitro fertilized pig blastocysts, NCSU23 culture medium was treated with various concentrations of LiCl (0, 0.01, 0.1, 1, and 10 mM) for 24 hours from day 6. As can be seen from Table 3, the blastocyst hatching rate cultured in the presence of LiCl significantly decreased dose-dependently. At the concentration of 10 mM LiCl supplementation in the culture medium, the blastocyst hatching rate was significantly lower than that of the other groups (3.1 ± 2.5%).

Ratio (n) Concentration Blastocyst count Extended blastocyst
Hatched blastocyst Control group 32 34.4 ± 2.2 ^a (11) 56.3 ± 5.0 ^a (18) 0.01 mM 33 48.5 ± 5.2 ^a (16) 36.4 ± 5.4 ^a (12) 0.1 mM 32 46.9 ± 8.5 ^ac (15) 28.1 ± 3.3 ^ac (9) 1.0 mM 34 61.8 ± 8.0 ^bc (21) 11.8 ± 7.3 ^bc (4) 10 mM 32 62.5 ± 11.1 ^bc (20) 3.1 ± 2.5 ^b (1)

Table 3 is a table showing the effect of LiCl on the hatching ability of pig blastocyst after 24 hours from day 6 at various concentrations in NCSU23 medium.

The other superscripts in the table, ^{a, b, and c,} are significantly different ( P <0.05).

We incubated blastocysts in the presence of LiCl for 48 hours from day 6. As can be seen in Figure 2a, the overall blastocyst hatching rate was still markedly different in a dose-dependent manner after 48 hours (Figure 2a). Compared to the control, the lowest blastocyst hatching rate was significant at 10 mM LiCl (65.6 ± 9.9 vs. 15.2 ± 7.3%).

We used confocal microscopy to investigate the intracellular location of the Dvl3 protein, one of three mammalian Dvl proteins, before and after hatching of porcine blastocysts. We observed that Dvl3 protein was located in the nucleus in expanded blastocysts before hatching. Interestingly, after incubation, the Dvl3 protein disappeared from the nucleus (FIG. 3). Large changes in intracellular location of Dvl3 indicate that levels of typical Wnt signaling decrease after hatching in porcine blastocysts.

For stimulation of Wnt signaling, increased plasma levels of free activating beta-catenin are stabilized and then migrated to the nucleus to activate the target gene of Wnt signaling. We evaluated the intracellular location of beta-catenin in dilated and hatched blastocysts by immunofluorescence. Similar to the nuclear migration of Dvl3, beta-catenin was strongly expressed in the nucleus of expanded blastocysts but they disappeared after incubation (FIG. 4).

These results thus strongly indicate that typical Wnt signaling is not activated after blastocyst hatching and Wnt signaling may be related to the regulation of blastocyst hatching in vitro.

In addition, in order to investigate the cause of the regulation of Wnt signaling on the blastocyst hatching rate in pigs, we determined the expression level of CDX2 mRNA, which plays a decisive role in TE differentiation and blastocyst formation, in Dkk1 in culture medium from day 6 for 48 hours. Or in each group of blastocysts cultured in vitro with LiCl (Barcroft LC, Hay-Schmidt A, Caveney A, Gilfoyle E, Overstrom EW, Hyttel P, Watson AJ. 1998. Journal of reproduction and fertility 114 (2): 327-339; Watson AJ, Barcroft LC. 2001. Regulation of blastocyst formation.Front Biosci 6: D708-730. Weitzman JB. 2005. Journal of biology 4 (1): 1).

Using RT-PCR, we observed that the expression level of CDX2 mRNA in the Dkk1 treated group was relatively high as compared to the control and LiCl treated groups (FIG. 6B), which was between Wnt signaling pathway and TE differentiation in blastocyst development. Indicates that there is a relationship.

In addition, to investigate whether Wnt signaling affects early blastocyst development, we treated initial blastocysts and loss embryos with Dkk1 or LiCl in the culture medium at the morula stage for 24 hours from day 5 and 7 Transfer to fresh medium without further treatment by day. The results showed that Dkk1 treated blastocysts were significantly more hatched compared to the control and contained more total cell numbers per blastocyst (FIG. 5A, B). In contrast, LiCl treated blastocysts had significantly lower hatching rates compared to controls and contained less total cell numbers per blastocyst (FIG. 5A, B). In addition, Dkk1 treated blastocysts showed a higher ratio of TE cells to ICM compared to control and LiCl treated blastocysts (FIG. 6A).

These data indicate that Wnt signaling affects TE differentiation in porcine blastocyst formation. Affect Give strong.

As can be seen from the present invention, it can be seen that Dickkopf-1 (Dkk1) increases porcine blastocyst hatching rate and TE differentiation.

1 shows the effect of Dkk1 on the hatching ability of porcine blastocysts. 1a shows blastocyst hatching rate after 24 and 48 hours from day 6 in the absence or presence of 10 ng / μl Dkk1 in NCSU23 medium, B-1 and B-2 of 1b expanded to photograph of porcine blastocyst at 48 hours after treatment Is 200 ×.
Figure 2 shows the effect of LiCl on the hatching ability of porcine blastocysts. 2a shows blastocyst hatching rate 24 hours and 48 hours after 6 days in the absence or presence of 10 mM LiCl in NCSU23 medium, and B-1 to B-5 of 2b were enlarged to photograph of porcine blastocyst at 48 hours after treatment. 200 ×.
Figure 3 shows immunofluorescence of porcine blastocysts with Dvl3 before and after incubation, with panel A: expanded blastocyst, panel B: hatched blastocyst, panel C: negative control without primary antibody Green: anti-Dvl3 FITC-attached Dyed, Red: PI Nuclear Dyed, Yellow: Merge.
4 is a diagram showing the immunofluorescence of β-catenin before and after hatching in porcine blastocysts. A, B, and C of FIG. 4 show immunostaining of β-catenin in expanded blastocysts before hatching. Represents immunostaining of β-catenin in hatched blastocysts after expansion (G, H, I in FIG. 4 represent controls). Green: anti-Dvl3 FITC-attached stain, red: PI nuclear stain.
5 is a diagram showing the change in incubation rate and total cell number according to the Wnt signaling control in the development of porcine blastocysts, 5a shows the post-development of each experimental group, 5b contrasted the change in hatching rate of blastocysts for each experimental group 5c is a bar graph comparing the change in total cell number after treatment of each experimental group.
FIG. 6 shows the correlation between Wnt signaling and TE differentiation in porcine blastocyst development. FIG. 6A shows the TE ratio to the total number of cells per blastocyst, and 6B shows the expression ratio of CDX2 mRNA, a TE-specific gene. Results are shown. In addition, 6C is a control group, 6D is a Dkk1 treatment group, 6E is a group stained by classifying the ICM (light blue) and TE (pink) of the LiCl treatment group.

The present invention will now be described in more detail by way of non-limiting examples. However, the following examples are described for the purpose of illustrating the present invention and the scope of the present invention is not to be construed as being limited by the following examples.

Example  1: oocyte acquisition Oocytes retrieval ) And in vitro maturity ( in vitro maturation  ; IVM )

Prepubertal pig ovaries were collected from the slaughterhouse and transferred to the laboratory and kept in saline at 30-37.8 ° C. Cumulus oocyte complexes (COCs) were aspirated from follicles (2-6 mm diameter) using a 10 ml syringe with 18 G needles. The COCs were washed three times with TL-HEPES medium containing 1 mg / ml BSA (low carbonate TALP; (Parrish et al. 1988)), 25 mM NaHCO ₃ , 10% (v / v) porcine follicular fluid, 0.57 mM cysteine, 0.22 μg / ml sodium pyruvate, 25 μg / ml gentamicin sulfate, 0.5 μg / ml p-FSH (Folltropin V; Vetrepharm, Ont., Canada), 1 μg / ml estradiol-17b, and 10 ng / ml In 50 groups of 500 ml Tissue Culture Medium 199 with Earle's salts (TCM-199; Gibco BRL, Grand Island, NY) supplemented with epidermal growth factor (EGF), matured under mineral oil for 42-44 hours under 5% CO ₂ conditions. (See Gupta MK, Uhm SJ, Lee HT. 2008a.Animal reproduction science 108 (1-2): 107-121.)

Example  2: in vitro fertilization ( In vitro Fertilization ; IVF )

In vitro fertilization of mature eggs was performed by Gupta MK, Uhm SJ, Lee HT. 2007b. Theriogenology 67 (2): 238-248. In summary, oocytes were washed three times with modified medium containing 1 mM caffeine sodium benzoate and 0.1% BSA (modified Tris-buffer medium) and placed in groups of 10 to 15 oocytes per 50 μl of modified medium. Pig testicles were collected from the slaughterhouse and brought to the laboratory and stored at 30 to 35.8 ° C. in 0.9% (w / v) saline supplemented with 75 μg / ml penicillin G and 50 μg / ml streptomycin sulfate. Spermatozoa were recovered from the myoblastomas in TL-HEPES and centrifuged at 800 rpm for 5 minutes to pelletize. The soft pellets were then subjected to a swim-up in Sp-TALP medium for 10 minutes. The supernatant was collected and washed twice by centrifugation at 800 rpm for 5 minutes. At the end of the washing process, sperm pellets were resuspended in fertilization medium and added to the fertilization droplet to obtain a final sperm concentration of 5 × 10 ⁵ sperm / ml. Sperm and oocytes were co-cultured for 6 hours in a 39.8 ° C., 5% CO 2 humidity environment prior to transfer to incubation medium in vitro for further culture.

Example  3: in vitro culture ( In vitro culture of embryos ; IVC )

Porcine embryos were cultured in NCSU23 medium supplemented with 0.4% BSA for 5 days and further cultured in NCSU23 medium supplemented with 10% (v / v) FBS for 2 days (Gupta MK, Uhm SJ, Lee SH, Lee HT. 2008b Molecular reproduction and development 75 (4): 588-597).

Treatment of the culture medium was treated with Dickkopf-1 (R & D systems, Minneapolis, MN) and lithium chloride (Sigma) for 24 hours and 24 hours on day 5 and day 6. The blastocysts were collected on days 7 and 8 for Hoechst 33342 staining, differential immunostaining, RT-PCR and immunohistochemistry for analysis of gene expression and incubation rate, total cell number, and TE / ICM ratio.

Example  4: Reverse transcriptase - Polymerase chain reaction RT - PCR )

For RT-PCR analysis, primers were designed for the sequence of each gene from Genebank (Table 1). PCR was performed in a total 20 μl volume using PCR Premix (AccuPower PCR Premix, Bioneer, Korea) according to the manufacturer's instructions. Initial denaturation was performed at 94 ° C. for 5 minutes followed by 35 cycles of PCR amplification. The amplification profile consisted of three steps: 30 seconds denaturation at 94 ° C., 30 seconds annealing at 56-60 ° C. (Table 1), 30 seconds extension at 72 ° C., depending on the primer pair used. After 35 amplification cycles, the samples were held at 72 ° C. for 7 minutes to involve full strand expansion and then specific PCR products were identified by clear EtBr-staining bands of sub-marine horizonal gel electrophoresis at 1% agarose. .

Gene GenBank Primer sequences Product size (bp) Annealing temp. (° C) CDX2 EU137688 Forward: 5'-GTCACCAGAGCTTCTCTGGG-3 '
Reverse: 5'-AGACCAACAACCCAAACAGC-3 ' 144 62 H2A BF703857.1 Forward: 5'- TGTCGTTGTCATGTCTGGTC-3 '
Reverse: 5'- CAACAGCTTGTTCAGCTCCT-3 ' 304 57

Table 1 shows the RT-PCR primers.

Example  5: for the analysis of nuclear numbers Blastocyst  Fluorescent dyeing

Nuclear staining for counting the number of neutrophils in blastocysts is described by Gupta MK, Uhm SJ, Han DW, Lee HT. 2007a. Molecular reproduction and development was performed as described in 74 (4): 435-444.

In summary, each blastocyst was fixed for 5 minutes in a fixed solution containing 2% formalin and 0.25% gluteraldehyde, placed on a glass slide, and stained for 10 minutes in a glycerol-based Hoechst 33342 (12.5 μg / ml) staining solution. Under UV irradiation with an epifluorescent microscope (Olympus, Tokyo, Japan) with a blue filter (excitation: 330-385 nm; emission: 420 nm; dichromatic: 400 nm), the total number of stained nuclei showing azure color was applied to each individual blastocyst. And digital images (Nikon Coolpix 990; Nikon Corporation, Tokyo, Japan) were taken.

Example  6: Immunofluorescence

For immunofluorescence, embryos were briefly exposed to pre-extraction solution (130 mM KCl, 25 mM HEPES (H6147, pH 6.9), 3 mM MgCl 2 and 0.06% Triton X-100) and 4% paraformaldehyde (PFA) and 0.5% Triton X-100 Involved fixation and infiltration in PBS containing. They were then overnight at 4 ° C. with 2% bovine serum albumin (BSA) and 2% skim milk powder in PBS 0.2% Tween-20 (PBST), followed by rabbit polyclonal anti-Dvl3 antibody (1:50; Cell signaling Technology). , Inc., USA) and rabbit polyclonal β-catenin antibody (1:50; Abcam, USA). Bound primary antibody was localized with FITC-attached anti-rabbit IgG secondary antibody (1: 250; Molecular Probes, Leiden, The Netherlands).

Embryos were then counterstained for 15 minutes with propidium iodide (200 μg / ml), a nuclear fluorophore, and mounted on slides of 10 μl antifade mounting medium (FluoroGuard ^™ , BIORAD, CA). Immunofluorescence labeling was analyzed at 633 nm with an epifluorescent microscope (Olympus, Olympus Corporation., Tokyo, Japan) with a confocal laser scanning microscope (FLUOVIEW FV1000-ASWv1.5, Tokyo, Japan) filter.

The experiment was repeated three times with each replicate for 5-8 embryos. Negative controls were performed by omitting the primary antibody and in all cases were unlabeled.

Example  7: TE  Discrimination for Rate Analysis Immunodiffusion

For ICM rate analysis, Kim NH, Uhm SJ, Ju JY, Lee HT, Chung KS. 1997. Some modifications to those described in Zygote (Cambridge, England) 5 (4): 365-370, blastocysts were differentially immunostained for ICM and TE on days 7 and 8.

In summary, blastocysts were removed from their zonal pelucida with 0.5% pronase and washed with TL-HEPES medium containing 0.1% PVP. The Jonah Felucida-free embryos were then exposed to whole rabbit anti-pig serum at 1: 5 dilution for 1 hour. They were then washed three times for 5 minutes and exposed to 1:10 dilution of Guinea Pig Complement containing 10 μg / ml propidium iodide (PI) and 10 μg / ml bisbenzimide for 1 hour. After a brief wash, the stained embryos were transferred to glass slides and examined under UV using an epifluorescent microscope. Blue and red cells were counted as cells from ICM and TE, respectively, and the TE rate was calculated as the number of red cells / blue and the total number of red cells X100.

All experiments of all the above examples were repeated at least four times and statistical analysis was performed using SAS software (Statistical Analysis System Inc., Cary, NC, USA). Embryonic development data were analyzed by Student t-test and the mean of cell number was compared by ANOVA followed by multiple pairwise comparisons after normal test (Kolmogorov-smirnov test with Lillie for calibration).

The data were presented as mean ± SEM and were judged to be significant when showing a difference of P ≦ 0.05.

<110> Konkuk University Industrial Cooperation Corp. <120> Composition for improving blastocyst hatching and trophectoderm          differentiation comprising Dickkopf-1 and method <160> 1 <170> Kopatentin 1.71 <210> 1 <211> 266 <212> PRT <213> Dkk-1 <400> 1 Met Met Ala Leu Gly Ala Ala Gly Ala Thr   1 5 10 15 Val Ala Ala Leu Gly Gly His Pro Leu Leu Gly Val Ser Ala Thr              20 25 30 Leu Asn Ser Val Leu Asn Ser Asn Ala Ile Lys Asn Leu Pro Pro Pro          35 40 45 Leu Gly Gly Ala Ala Gly His Pro Gly Ser Ala Val Ser Ala Ala Pro      50 55 60 Gly Ile Leu Tyr Pro Gly Gly Asn Lys Tyr Gln Thr Ile Asp Asn Tyr  65 70 75 80 Gln Pro Tyr Pro Cys Ala Glu Asp Glu Glu Cys Gly Thr Asp Glu Tyr                  85 90 95 Cys Ala Ser Pro Thr Arg Gly Gly Asp Ala Gly Val Gln Ile Cys Leu             100 105 110 Ala Cys Arg Lys Arg Arg Lys Arg Cys Met Arg His Ala Met Cys Cys         115 120 125 Pro Gly Asn Tyr Cys Lys Asn Gly Ile Cys Val Ser Ser Asp Gln Asn     130 135 140 His Phe Arg Gly Glu Ile Glu Glu Thr Ile Thr Glu Ser Phe Gly Asn 145 150 155 160 Asp His Ser Thr Leu Asp Gly Tyr Ser Arg Arg Thr Thr Leu Ser Ser                 165 170 175 Lys Met Tyr His Thr Lys Gly Gln Glu Gly Ser Val Cys Leu Arg Ser             180 185 190 Ser Asp Cys Ala Ser Gly Leu Cys Cys Ala Arg His Phe Trp Ser Lys         195 200 205 Ile Cys Lys Pro Val Leu Lys Glu Gly Gln Val Cys Thr Lys His Arg     210 215 220 Arg Lys Gly Ser Gly Leu Glu Ile Phe Gln Arg Cys Tyr Cys Gly 225 230 235 240 Glu Gly Leu Ser Cys Arg Ile Gln Lys Asp His His Gln Ala Ser Asn                 245 250 255 Ser Ser Arg Leu His Thr Cys Gln Arg His             260 265

Claims (7)

delete delete delete A method of improving the blastocyst hatching rate of porcine embryos, comprising: culturing pig embryos in a culture medium, and treating the blastocyst hatching ratio of pig embryos comprising the step of treating Dickkopf-1 to the culture solution for 24 to 48 hours on the 6th day of culture. How to improve. The method of claim 4, wherein the Dickkopf-1 is treated at a concentration of 10 ng / ul at 0.1 ng / ul. delete delete

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