JP2024004773A - In-vitro culture method of bovine embryo, bovine embryo, and method for producing bovine - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an in-vitro culture method of a bovine embryo in which rapid regression or loss of differentiation potency after an embryo blastocyst stage is suppressed, an in-vitro cultured embryo having gene expression and morphological features similar to a live embryo is obtained, and a bovine embryo using the in-vitro culture method of bovine embryo, and a method for producing bovine.

SOLUTION: An in-vitro culture method of a bovine embryo includes a culture process of culturing an embryo in a bovine blastocyst stage on gel in which culture medium is impregnated. A bovine embryo is obtained by the in-vitro culture method of a bovine embryo. In a bovine embryo obtained by in-vitro fertilization, one or more genes selected from the group consisting of IFNT, GJB1, MYL3, ETS2, ID2, SSLP1 and ASCL2 are expressed in 10 days from in-vitro fertilization. A method for producing bovine includes a transplanting process of transplanting a bovine embryo, which is obtained by the in-vitro culture method of a bovine embryo, into a recipient bovine to allow it to conceive.

SELECTED DRAWING: None

COPYRIGHT: (C)2024,JPO&INPIT

Description

Application for application of Article 30, Paragraph 2 of the Patent Act (1) Publication date: September 27, 2021, Publication: THE FASEB JOURNAL, Vol. 35, Issue 10, e21904, (2) Website publication date: October 14, 2021, Website address: https://www. hokudai. ac. jp/news/2021/10/post-918. html; https://www. hokudai. ac. jp/news/pdf/211014_pr2. pdf, (3) Publication date: November 5, 2021, Publication: Nikkei Sangyo Shimbun, Morning edition dated November 5, 2021, page 7

The present invention relates to a method for culturing bovine embryos in vitro, bovine embryos, and a method for producing bovines.

Early mammalian embryo culture technology focuses on development up to the blastocyst stage, which can develop independently of the mother's body (uterus). Early mammalian embryos can be developed in cell culture medium until implantation in the uterus, and are generally used for in vitro culture of early embryos in laboratory animals such as mice, humans, and livestock such as cows for research, industry, and research. Widely practiced for medical purposes. By combining in vitro fertilization (in vitro fertilization) and in vitro culture, the embryos that have developed to the blastocyst stage are transferred to fertilized eggs and returned to the uterus for ontogeny, making it possible to artificially control reproduction.

The developmental stage of the embryo that implants in the uterus is species-specific; in mice and humans, the embryo implants immediately after the blastocyst stage, but in cows, it does not implant at the blastocyst stage, and instead develops into an oval embryo and an elongated embryo. It is known that implantation occurs after development into an embryo and then into a string stage embryo. Therefore, in theory, it can be said that bovine embryos can develop autonomously even after the blastocyst stage without implanting in the uterus. However, although in vitro culture systems for developing bovine embryos after the blastocyst stage have been attempted around the world, there has been no research to date that has proven development biologically and has succeeded in producing individuals. .

Bovine embryos grow rapidly with an exponential increase in cell number after the blastocyst stage. In recent years, methods have been sought to dramatically increase the efficiency of breeding and improving cattle by estimating the genetic potential of cattle by biopsying a portion of fertilized eggs, but the number of cells in a blastocyst stage embryo is There are concerns that the fertility rate of embryos damaged by biopsy will decrease. However, if a system that allows for in vitro culturing of bovine embryos after the blastocyst stage is created, the proportion of damage caused by biopsy to the whole embryo will be relatively reduced, and the implantation potential of early embryos will be maintained while maintaining genetic potential. It is possible to produce bovine embryos and individuals that have been evaluated.

Most attempts to develop cattle to the blastocyst stage or later can be roughly divided into two approaches: eutrophication of culture medium components and embedding the embryo in a matrix such as gel (for example, see Non-Patent Document 1). be done.

Brandao D O et al., “Post hatching development: a novel system for extended in vitro culture of bovine embryos.”, Biol Reprod Vol. 71, Issue 6, pp. 2048-2055, 2004.

However, in the conventional in vitro culture method for bovine embryos as typified by Non-Patent Document 1, although it is possible to extend the survival period by several days, the characteristics of the embryo such as differentiation potential are lost during culture. Furthermore, previous reports have not verified the expression of marker genes or the developmental potential after transplantation into the mother, and it has not been possible to discuss the embryological similarities with living embryos.

The present invention has been made in view of the above circumstances, and is an in vitro embryo that suppresses rapid degeneration and loss of differentiation ability of embryos after the blastocyst stage, and has gene expression and morphological characteristics similar to living embryos. Provided are a method for culturing bovine embryos in vitro that yields cultured embryos, and a method for producing bovine embryos and cows using the method for culturing bovine embryos in vitro.

As a result of extensive research to achieve the above objective, the inventors have found that by culturing bovine blastocyst-stage embryos in vitro on gel impregnated with a medium, rapid growth of embryos beyond the blastocyst stage can be achieved. The present inventors have discovered that degeneration and loss of differentiation ability are suppressed, and that bovine embryos can be obtained that have gene expression and morphological characteristics similar to those of living embryos, and have completed the present invention.

That is, the present invention includes the following aspects.
(1) A method for culturing bovine embryos in vitro, which includes a culturing step of culturing bovine blastocyst-stage embryos on a gel impregnated with a medium.
(2) The method for culturing bovine embryos in vitro according to (1), wherein in the culturing step, the culturing is carried out in the presence of oxygen of 3 v/v % or more and 7 v/v % or less.
(3) The method for culturing bovine embryos in vitro according to (1) or (2), wherein in the culturing step, the embryos are cultured for one day or more.
(4) The in vitro culture of bovine embryos according to any one of (1) to (3), wherein the medium contains a serum substitute of 10 v/v % or more and 50 v/v % or less based on the volume of the medium. Method.
(5) The method for culturing bovine embryos in vitro according to any one of (1) to (4), wherein the gel is an agarose gel.
(6) A bovine embryo obtained by the in vitro culture method for bovine embryos according to any one of (1) to (5).
(7) A bovine embryo obtained by in vitro fertilization,
A bovine embryo in which one or more genes selected from the group consisting of IFNT, GJB1, MYL3, ETS2, ID2, SSLP1, and ASCL2 are expressed 10 days after in vitro fertilization.
(8) Ten days after in vitro fertilization, the expression level of one or more genes selected from the group consisting of OCT4, NANOG, and SOX2 is 1/10 or less of the expression level of the same gene 8 days after in vitro fertilization. , the bovine embryo described in (7).
(9) A method for producing cattle, which includes a step of transplanting a bovine embryo obtained by the in vitro culture method for bovine embryos according to any one of (1) to (5) into an embryonic cow to impregnate it.

According to the in vitro culture method for bovine embryos of the above aspect, rapid degeneration and loss of differentiation ability of embryos after the blastocyst stage are suppressed, and in vitro cultured embryos having gene expression and morphological characteristics similar to living embryos are produced. can get. The bovine embryo of the above embodiment has gene expression and morphological characteristics similar to those of a living embryo, in which rapid degeneration and loss of differentiation ability of the embryo after the blastocyst stage are suppressed. The method for producing cattle according to the above aspect uses a bovine embryo obtained by the above-mentioned in vitro culture method for bovine embryos, and can contribute to the breeding and improvement of desired cattle.

FIG. 1 is a schematic configuration diagram showing an example of an in vitro culture system for bovine embryos used in the in vitro culture method for bovine embryos according to the present embodiment. 1 is a schematic configuration diagram showing an on-gel culture method using a bovine blastocyst stage embryo produced by a conventional droplet culture method in Example 1. FIG. This is an ultrasound image of a 68-day-old embryo that was artificially inseminated and cultured on-gel in Example 1. 1 is an observation image of sections of a D12 in vivo-derived embryo and a D14 on-gel cultured embryo in Example 1. The scale bar indicates 200 μm. Using anti-SOX2 antibodies and anti-SOX17 antibodies from D12 on-gel cultured embryos cultured under 21% (v/v) or 5% (v/v) O ₂ in Example 1, and D12 in vivo-derived embryos. This is a confocal image of fluorescent immunostaining. The scale bar indicates 200 μm. 1 is a confocal image of fluorescent immunostaining of on-gel cultured embryos at D8.5, D9.0, and D9.5 in Example 1 using an anti-OCT4 antibody and an anti-CDX2 antibody. The scale bar indicates 200 μm. 1 is a plot showing the ratio of CDX2 and OCT4 double positive cells to the total number of CDX2 positive cells in on-gel cultured embryos in Example 1. 1 is a confocal image of fluorescent immunostaining of on-gel cultured embryos at D8.5, D9.0, and D9.5 in Example 1 using an anti-SOX2 antibody and an anti-SOX17 antibody. The scale bar indicates 200 μm. 1 is a confocal image of fluorescent immunostaining using an anti-SOX2 antibody, an anti-SOX17 antibody, and an anti-NANOG antibody of an in vivo-derived embryo at D10 in Example 1. The scale bar indicates 200 μm. 1 is a graph showing the ratio of SOX2-positive or negative SOX17-positive cells to the total number of SOX17-positive cells in on-gel cultured embryos at D8.0, D9.0, and D10 in Example 1. 1 is a confocal image of fluorescent immunostaining using an anti-SOX2 antibody and an anti-SOX17 antibody of an on-gel cultured embryo treated with DMSO or 100 μM of ROCK inhibitor Y27632 in Example 1. The scale bar indicates 200 μm. 1 is a graph comparing the expression levels of pluripotency-related genes in cell samples of D8.0 IVC embryos and D14 in vivo-derived embryos in Example 1 based on RNA-seq. Asterisks represent significant differences (p<0.05) analyzed using edgeR test. A self-organizing map based on the expression level of transcription factors calculated by the RPKM value of each transcript from the RNA-seq data of cell samples of IVC embryos at D8.0 and in vivo-derived embryos at D14 in Example 1. be. 1 is a graph showing the results of a hypergeometric distribution test (top) and gene set enrichment analysis (bottom) of gene ontology (GO) analysis in Example 1. The top 5 GO terms for each test are displayed. Asterisks represent significant differences (p<0.05). 1 is a heat map based on RPKM values of genes related to Wnt, FGF, and VEGF signaling pathways in Example 1. Asterisks represent significant differences (p<0.05). FIG. 2 is a transmission electron microscopy (TEM) image showing gap junctions of trophoblast cells (left) in an on-gel cultured embryo at D20 and binucleated cells (right) in an on-gel cultured embryo at D21 in Example 1. FIG. 1 is a graph showing the results of RT-qPCR using TE cells of on-gel cultured embryos at D8.0, D10, and D14 in Example 1. "N.D." represents not detected under the threshold of 50 cycles. Asterisks represent significant differences, "*" p<0.05, "**" p<0.01. 1 is a graph showing the results of RT-qPCR using TE cells of D10 on-gel cultured embryos and D10 in vivo-derived embryos in Example 1. "N.D." represents not detected under the threshold of 50 cycles. Asterisks represent significant differences, "*" p<0.05, "**" p<0.01. An image showing a calf born vaginally on September 21, 2021 in Example 2 (top left) and an image showing that the subsequent birth was morphologically normal (bottom left), and 2021 Image (top right) showing a calf born vaginally on November 29th.

≪In vitro culture method for bovine embryos≫
The in vitro culture method for bovine embryos of this embodiment includes a culturing step of culturing a bovine blastocyst stage embryo on a gel impregnated with a medium.

The in vitro culture method for bovine embryos of this embodiment differs from conventional in vitro culture methods, such as methods that rely on adhesion to scaffolds (substrates such as feeder cells or plastic petri dishes) or embedding them in gel. This is a very simple method of placing the embryo on a gel. The in vitro culture method for bovine embryos of the present embodiment has the advantage that, in addition to not requiring any special techniques for application, the embryos can be easily handled after culture. The bovine embryo obtained by the method for in vitro culturing of bovine embryos of this embodiment can be directly manipulated with a glass capillary or micropipettor without undergoing steps such as detachment from the substrate or destruction of the culture container. Therefore, physical damage to embryo cells and contamination with substrates during manipulation are extremely small, improving the reliability of the biopsy and leading to smooth embryo transfer. Growth is also good.

Furthermore, the most innovative point in the in vitro culture method for bovine embryos of this embodiment is that the embryos can be cultured while being exposed to the gas phase. In the so-called "tunnel method," in which embryos are cultured in a cavity created within a conventional gel, the entire embryo comes into contact with the gel as the embryo increases in cell number and volume. This is thought to impede efficient gas exchange that supports the survival of Epi cells, and in fact, significant loss of Epi cells due to necrosis has been observed.

On the other hand, the in vitro culture method for bovine embryos according to the present embodiment is based on the idea of exposing the embryo to two phases, a gas phase and a liquid phase, and the method is based on the idea that the embryo is exposed to two phases: a gas phase and a liquid phase, and nutrients are supplied from the liquid phase. This is an innovative method that simultaneously achieves efficient gas exchange through contact with the gas phase. The in vitro culture method for bovine embryos of the present embodiment is the first application of the "gas phase-liquid phase interface culture method", which is one of the classical culture methods, to early embryo culture. In the general gas-liquid interface culture method, cells are seeded on a so-called "insert" such as a porous membrane, and this is placed on an assay medium, allowing exposure to two layers of cells. do. In the case of bovine embryos, rapid differentiation occurs when they adhere to the substrate, making it difficult to maintain their embryonic properties.In order to avoid this, the in vitro culture method for bovine embryos of this embodiment uses a gel instead of an insert. This solved the above problem.

That is, according to the in vitro culture method for bovine embryos of the present embodiment, having the above structure suppresses rapid degeneration and loss of differentiation ability of the embryo after the blastocyst stage, and achieves gene expression similar to that of living embryos. An in vitro cultured embryo (preferably a mature blastocyst stage embryo or later) having the following morphological characteristics is obtained.

Next, each step of the in vitro culture method for bovine embryos of this embodiment will be described in detail below.

<Culture process>
In the culturing step, bovine blastocyst-stage embryos are cultured on a gel impregnated with a medium.

FIG. 1 is a schematic diagram showing an example of an in vitro culture system for bovine embryos used in the in vitro culture method for bovine embryos according to the present embodiment. This process will be described below with reference to FIG.

In the in vitro culture system 10 for bovine embryos, a gel 1 is arranged in each well of a culture container 3, and a bovine blastocyst stage embryo B is arranged on the gel 1. The medium 2 may be in any state as long as it is impregnated with the gel 1, but as shown in FIG. good. Thereby, drying of the gel 1 can be prevented, and nutritional components contained in the medium 2 can be continuously supplied to the bovine blastocyst stage embryo B.

In addition, in the in vitro culture system 10 for bovine embryos shown in FIG. 1, the bovine blastocyst-stage embryo B is cultured on the gel 1 impregnated with the medium 2, so that it is separated into two phases: a gas phase and a liquid phase. Can be cultured while being exposed.

[gel]
As the gel used in the culturing process, known gels commonly used in culturing cells or cell aggregates can be used. Materials constituting such a gel include, for example, extracellular matrix-derived components that gel, polysaccharides, chitin, poly(3-hydroxyalkanoate) (poly(β-hydroxybutyrate)), and poly(3-hydroxyalkanoate) (poly(β-hydroxybutyrate)). octanoate), poly(3-hydroxy fatty acid), fibrin, agar, agarose, etc., but are not limited to these. Examples of polysaccharides include alginate, cellulose, dextran, pullulane, polyhyaluronic acid, and derivatives thereof.

Extracellular matrix-derived components to be gelled include, for example, collagen (type I, type II, type III, type V, type XI, etc.), mouse EHS tumor extract (type IV collagen, laminin, heparan sulfate proteoglycan, etc.) ) reconstituted basement membrane components (trade name: Matrigel), glycosaminoglycans, hyaluronic acid, proteoglycans, gelatin, etc., but are not limited to these.

These can be used alone or in combination of two or more.
Among these, it is preferable to use a highly transparent gel, and agarose gel is more preferable, since it can be clearly observed with the naked eye or with an inverted microscope.

[Culture medium]
The medium used in the culture process is preferably a completely synthetic medium that does not contain unknown components such as serum, and is suitable for stem cell development because it can suppress rapid differentiation of the embryo and support development beyond the blastocyst stage. More preferably, it contains a serum substitute that is also used for culture. When the medium contains a serum substitute, the viability and integrity of Epi cells, which are sensitive to the external environment such as culture conditions, can be dramatically improved, and long-term culture becomes possible.

Serum substitutes include, for example, albumin (e.g. lipid-rich albumin, albumin substitutes such as recombinant albumin; vegetable starches, dextran and protein hydrolysates, etc.), transferrin (or other iron transporters), fatty acids, insulin. , collagen precursors, trace elements, 2-mercaptoethanol, 3'-thioglycerol, and equivalents thereof. Further examples include KnockOut (registered trademark) Serum Replacement (KSR), GlutaMax (registered trademark), and the like. These can be used alone or in combination of two or more. Among these, KSR is preferred as a serum substitute.

The concentration of the serum substitute (preferably KSR) in the medium can be 1 v/v% or more, preferably 10 v/v% or more and 50 v/v% or less, and 15 v/v% or more and 45 v/v% or less. is more preferable, more preferably 20 v/v% or more and 40 v/v% or less, particularly preferably 25 v/v% or more and 35 v/v% or less.

Examples of the basal medium used in the culture process include αMEM medium, Neurobasal medium, Neural Progenitor Basal medium, NS-A medium, BME medium, BGJb medium, CMRL 1066 medium, minimum essential medium (MEM), Eagle MEM, and Dulbecco's modified medium. Eagle medium (DMEM), Glasgow MEM (GMEM), Improved MEM Zinc Option, IMDM, Medium 199 medium, DMEM/F12 medium, StemPro-34SFM medium, Ham's medium, RPMI 1640 medium, HTF medium, Fischer' s medium, Advanced DMEM , Advanced DMEM/F12, Advanced MEM, Advanced RPMI medium, a mixed medium thereof, and the like, but are not limited to these. Among them, RPMI 1640 medium is preferred.

The medium may also contain other known additives. Additives are not particularly limited, but include, for example, polyamines, minerals, sugars (e.g., glucose), organic acids (e.g., pyruvic acid, lactic acid, etc.) and their salts (sodium salts, potassium salts, etc.), amino acids (e.g., essential amino acids (EAA), non-essential amino acids (NEAA), L-glutamine, etc.), reducing agents (e.g., 2-mercaptoethanol, etc.), vitamins (e.g., ascorbic acid, d-biotin, etc.), antibiotics (e.g., Streptomycin, penicillin, etc.), buffers (eg, HEPES, etc.), nutritional additives (eg, B27 supplement, N2 supplement, StemPro-Nutrient Supplement, etc.), and selenium compounds (sodium selenite, etc.). These can be used alone or in combination of two or more. Each additive can be included in known concentration ranges. On the other hand, since growth factors such as fibroblast growth factor (FGF) are known to induce cell differentiation, it is preferable that the medium does not contain growth factors or growth factors. Furthermore, since steroids also exhibit estrogen-like effects and may affect survival and cell differentiation, it is preferable that the medium does not contain steroids.

[Culture conditions]
In the culturing process, for example, the bovine blastocyst stage embryo, the gel, and the medium can be cultured in a known cell culture container in an arrangement as shown in FIG. 1.

The culture conditions are not limited to the following, but since it is close to the in utero environment, it is preferably carried out in the presence of oxygen of 3 v/v% or more and 7 v/v% or less, more preferably 4 v/v% or more and 6 v/v% or less. Can be done. Further, the oxygen concentration is within the above range, and the carbon dioxide is 1 v/v% or more and 10 v/v% or less, preferably 3 v/v% or more and 7 v/v% or less, and more preferably 4 v/v% or more and 6 v/v% or less. It can be carried out in the presence of carbon.

The culture temperature is about 30°C or higher and 40°C or lower, preferably about 38.5°C.

The culture period is preferably one day or more, more preferably about two days (eg, 48±12 hours, preferably 48±6 hours). Alternatively, it may be for a long period of two days or more.

In the method for culturing bovine embryos of the present embodiment, it is confirmed that a mature blastocyst stage embryo or later embryo has been obtained by visual observation or morphological observation using a microscope, or by analyzing the expression of marker genes described below. be able to.

[Method for producing bovine blastocyst stage embryo]
The bovine blastocyst stage embryo used in the culture step may be extracted from the bovine uterus or may be obtained by in vitro culture, but is preferably obtained by in vitro culture. Bovine blastocyst-stage embryos produced by in vitro culture are described, for example, in Reference 1 (Brinster RL et al., “A METHOD FOR IN VITRO CULTIVATION OF MOUSE OVA FROM TWO-CELL TO BLASTOCYST.”, Exp Cell Res., Vol. 32, pp. 205-208, 1963.) and others, it can be induced from fertilized eggs that have been fertilized in vitro. Or, for example, reference 2 (Holm P et al., “High bovine blastocyst development in a static in vitro production system using SOFaa medium supplemented with sodium citrate and myo-inositol with or without serum-proteins”, Theriogenology, Vol. 52 It can also be derived from fertilized eggs that have been fertilized in vitro using the in vitro culture method described in , Issue 4, pp. 683-700, 1999.

Specifically, the method for producing a bovine blastocyst stage embryo by in vitro culture can include the following steps.
Step 1 of obtaining a fertilized egg by in vitro fertilization of oocytes and sperm; and
Step 2 of culturing the obtained fertilized eggs in microdroplets of a medium.

(Step 1)
The oocytes used in step 1 are collected from the ovaries of cows. For example, a cumulus cell-oocyte complex (COC) is collected from the ovary of a cow, and the COC is heated at a temperature of about 30° C. to 40° C. (preferably about 38.5° C.) and 1 v/v% to 10 v/v%. v% or less (preferably 3 v/v% or more and 7 v/v% or less, more preferably 4 v/v% or more and 6 v/v% or less), and in a humidified atmosphere of about 80 RH% or more (preferably , under saturated water vapor of approximately 100 RH%). Examples of the medium used for culturing COC include, but are not limited to, TCM-199 medium (manufactured by Thermo Fisher Scientific).

Furthermore, the sperm used in step 1 is collected from bulls by a known method. Alternatively, cryopreserved semen can be thawed and used.
Examples of the culture medium used during in vitro fertilization include, but are not limited to, bracket and oliphant (BO) media. The BO medium contains about 1.0 mmol/L to 4.0 mmol/L (preferably about 2.5 mmol/L) theophylline, about 6.0 μg/mL to 9.0 μg/mL (preferably about 7 .5 μg/mL) of heparin sodium salt. The semen is diluted with BO medium and the sperm concentration is adjusted accordingly.

Next, in step 1, a micro droplet (about 100 μL) of BO medium containing sperm is prepared on a known culture container, and mature oocytes (or COCs) are added to the micro droplet. Thereafter, the temperature is about 30°C or more and 40°C or less (preferably about 38.5°C), 1v/v% or more and 10v/v% or less (preferably 3v/v% or more and 7v/v% or less, more preferably 4v/v%). Fertilized eggs are obtained by insemination in the presence of carbon dioxide (6 v/v% or less) and in a humidified atmosphere of about 80 RH% or more (preferably about 100 RH%).

The insemination time in step 1 is preferably 10 hours or more, more preferably 12 hours or more.

(Step 2)
In step 2, the fertilized eggs obtained in step 1 are cultured in microdroplets (about 100 μL) of the medium. Examples of the medium used in step 2 include, but are not limited to, mSOFai medium. When COC is used, cumulus cells can be removed by transferring the fertilized eggs obtained in step 1 to mSOFai medium and pipetting with a glass pipette or the like.

The culture conditions in step 2 are adjusted so that each microdroplet (approximately 100 μL) contains about 10 to 30 fertilized eggs (preferably 20 fertilized eggs), and the culture conditions are adjusted to about 30°C to 40°C. ℃ or less (preferably about 38.5°C), 1 v/v% or more and 10 v/v% or less (preferably 3 v/v% or more and 7 v/v% or less, more preferably 4 v/v% or more and 6 v/v% or less) By culturing in the presence of carbon dioxide and a humidified atmosphere of about 80 RH% or more (preferably about 100 RH%), a bovine blastocyst stage embryo is obtained.

The culture period is preferably 3 days or more, more preferably 5 days or more, and even more preferably 8 days (for example, 96±12 hours, preferably 96±6 hours).

The fact that a bovine blastocyst stage embryo has been obtained can be confirmed by observing the morphology of the embryo visually or under a microscope.

≪Bovine embryo≫
<First embodiment>
The bovine embryo according to the first embodiment of the present invention is obtained by the above-described in vitro culture method for bovine embryos.

As shown in the examples below, the bovine embryo of this embodiment is a mature blastocyst-stage embryo, unlike the blastocyst-stage embryo obtained by conventional in vitro culture methods, and is a mature blastocyst-stage embryo. Rapid degeneration and loss of differentiation potential of the embryo are suppressed, and the embryo has gene expression and morphological characteristics similar to living embryos. In addition, as shown in the Examples described below, the bovine embryo of this embodiment has been confirmed to have the ability to implant when transplanted into a recipient cow, and furthermore, it has been confirmed that the bovine embryo derived from the bovine embryo has the ability to implant. It has been confirmed that it has ontogenic potential since it was delivered vaginally in embryonated cows. However, due to differences in gene expression profiles, there is a difference between bovine embryos derived from living organisms (especially mature blastocyst stage embryos) and other bovine embryos obtained by conventional in vitro culture methods (especially blastocyst stage embryos). Identifying the differences and identifying the bovine embryo of this embodiment is very difficult and impractical. Therefore, it is practical to specify it by the manufacturing method.

<Second embodiment>
The bovine embryo according to the second embodiment of the present invention is a bovine embryo obtained by in vitro fertilization, and is selected from the group consisting of IFN, GJB1, MYL3, ETS2, ID2, SSLP1, and ASCL2 10 days after in vitro fertilization. One or more genes associated with the disease are expressed.

IFNT is a gene encoding interferon tau, a pregnancy recognition factor. It is produced and secreted in trophoblast cells and has a period-specific expression pattern from the blastocyst stage to the time of implantation.
GJB1 is a gene encoding gap junction protein beta 1 (gap junction protein beta 1) protein, which is a transmembrane protein.
MYL3 is a gene encoding myosin light chain 3.
ETS2 is a gene encoding ETS proto-oncogene 2, which is a transcription factor presumed to exist upstream of IFNT.
ID2 is a gene encoding differentiation inhibitory protein 2, which is a transcriptional regulatory factor.
SSLP1 is a gene encoding the secreted seminal vesicle Ly6 protein, which is a member of the Ly-6/uPAR superfamily. SSLP1 is specifically expressed in binucleate cells, which are typical of bovine embryonic trophoblast cells.
ASCL2 is a gene encoding achaete scute-like 2, a Wnt target transcription factor.
That is, the above-mentioned genes are marker genes whose expression increases after the mature blastocyst stage embryo. Since one or more genes among these genes are expressed 10 days after in vitro fertilization, the bovine embryo of this embodiment is a mature blastocyst stage embryo or later embryo 10 days after in vitro fertilization. It can be said that it forms a

In addition, in the bovine embryo of this embodiment, the expression level of one or more genes selected from the group consisting of OCT4, NANOG, and SOX2 10 days after in vitro fertilization is different from the expression level of the same gene 8 days after in vitro fertilization. , preferably 1/10 or less, more preferably 1/100 or less. More preferably, 10 days after in vitro fertilization, the expression level of one or more of the above-mentioned genes is below the lower measurement limit, that is, the gene is not expressed, or the expression can hardly be confirmed.
That is, the above-mentioned genes are typical pluripotency-related genes, and all of them are marker genes whose expression decreases or disappears after the mature blastocyst stage embryo. 10 days after in vitro fertilization, the expression level of one or more of these genes is lower than the expression level of the same gene 8 days after in vitro fertilization. Ten days after fertilization, it can be said that a mature blastocyst stage embryo or later embryo has been formed.

The expression level of the marker gene described above can be confirmed by analysis using known methods such as immunostaining, FACS analysis, quantitative PCR, and RNAseq.

The expression profile of the above genes in the bovine embryo of this embodiment is similar to that in a living embryo. On the other hand, as shown in Examples (particularly FIG. 2C) to be described later, in living embryos, the hypoblast is formed and clearly visible 12 days after fertilization, whereas in this embodiment, the hypoblast is formed and clearly visible. In bovine embryos, the hypoblast is formed 14 days after in vitro fertilization, and the formation of the hypoblast is delayed.

In addition, when the bovine embryo of this embodiment is transplanted into a recipient cow, it has implantation ability, and furthermore, a calf derived from the bovine embryo can be delivered vaginally in the recipient cow. The bovine embryo has ontogenic potential.

The method for producing the bovine embryo of this embodiment is not particularly limited, but it can be obtained, for example, by the above-mentioned in vitro culture method for bovine embryos.

≪Cow production method≫
The method for producing cattle of the present embodiment includes a step of transplanting a bovine embryo obtained by the above-described in vitro culture method of bovine embryos into a recipient cow to impregnate the cow.

According to the cow production method of the present embodiment, desired cows can be easily bred and improved by using bovine embryos obtained by the above-described in vitro culture method for bovine embryos.

Next, the steps constituting the cow production method of this embodiment will be described in detail below.

<Transplanting process>
In the transplantation step, the bovine embryo obtained by the above-described in vitro culture method for bovine embryos is transplanted into a recipient cow to impregnate it.

Preferably, the age of the bovine embryo to be transferred and the ovulation cycle of the recipient cow are synchronized. The ovulation cycle can be regulated by administering hormones such as estradiol, gonadotropin-releasing hormone GnRH, or prostaglandin _F2α to the recipient cow. Further, before the synchronization of the ovulation cycle, the ovulation cycle and the inside of the uterus of the embryonic cow may be examined in advance using an ultrasonic examination device or the like.
The drugs used to synchronize the ovulation cycle and the bovine embryo can be administered to the recipient cow using, for example, an intravaginal drug release device (CIDR).

<Sorting process>
The bovine production method of the present embodiment can further include, before the transplantation step, a selection step of selecting bovine embryos obtained by the above-described in vitro culture method for bovine embryos.

Immature blastocyst-stage embryos obtained by conventional in vitro culture methods have a small number of cells, and the maximum number of cells that can be used for testing is about 15. Therefore, it was not possible to collect a sufficient amount of DNA samples, making it difficult to conduct multiple genetic diagnoses. Alternatively, in order to perform multiple genetic diagnoses, DNA amplification is essential, which poses a problem in accuracy.

On the other hand, the bovine embryo obtained by the above-mentioned in vitro culture method has a larger number of cells than the immature blastocyst stage embryo obtained by the conventional in vitro culture method, so it is difficult to collect a sufficient amount of DNA sample. It becomes possible. As a result, multiple diagnoses and analyzes such as determining the sex of bovine embryos, various genetic diagnoses, and analyzing DNA markers such as meat quality become possible. Furthermore, since DNA amplification is not required to perform multiple genetic diagnoses, highly accurate testing is possible in one step.

Based on the above, in the selection process, a sufficient amount of DNA samples are collected from bovine embryos, various genetic diagnoses, etc. and DNA marker analysis are performed, and based on the results, bovine embryos with desired traits are selected. , can be used in the above-mentioned transplantation step.

EXAMPLES Hereinafter, the present invention will be explained with reference to examples, but the present invention is not limited to the following examples.

[Example 1]
With the aim of elucidating the differentiation mechanism underlying the lineage separation of primary and secondary cells that mediate organized embryonic development in cattle, we used a novel culture system named "on-gel culture" to Cell lineage segregation in bovine embryos was evaluated.

(Raw materials and methods)
1. Preparation of bovine embryos Collection of bovine oocytes, in vitro oocyte maturation, fertilization (IVF), and subsequent in vitro embryo culture up to the blastocyst stage were performed as previously reported. Specifically, cumulus cell-oocyte complexes (COCs) collected from slaughterhouse-derived ovaries were grown in TCM-199 medium (Thermo Fisher Scientific Inc.) at 38.5°C in air with 5% CO2 _. The cells were cultured for 20 to 22 hours under saturated water vapor pressure. Oocytes matured in vitro were transferred to Brackett and Oliphant (BO) medium containing 2.5 mM theophylline (Wako Pure Chemical Industries, Ltd.) and 7.5 μg/mL heparin sodium salt (Nacalai Tesque Co., Ltd.). Subsequently, the frozen and thawed semen was centrifuged and washed at 600×g for 7 minutes in BO medium. Sperm were added to the droplets containing COC to a final concentration of 5×10 ⁶ cells/mL. Fertilized eggs were incubated in mSOFai medium under saturated water vapor with 5% (v/v) CO ₂ and 5% (v/v) O ₂ by removing the cumulus cells around the eggs by pipetting after 12 h incubation. , and cultured at 38.5°C for 8 days.

Embryos grown in vivo (living embryos) were non-surgically perfused with warmed Lactlinger solution (Nippon Zenyaku Kogyo Co., Ltd.) on days 10, 12, and 14 after artificial insemination of estrus-synchronized cows. Collected by.

2. Library preparation for RNA-seq 14 days from in vitro fertilization (IVF D8) blastocysts and in vivo grown artificial insemination using a microfeather blade (Feather Safety Razor Co., Ltd.) under a stereomicroscope. The inner cell mass (ICM) and the scutellum were mechanically removed from each day-old embryo (in vivo D14 elongated embryo). Total RNA of the remaining trophectoderm (TE) samples was extracted using the ReliaPrep ^™ RNA Cell Miniprep System (Promega) according to the manufacturer's protocol.

TE mechanically isolated from 20 embryos from D8 blastocyst stage embryos and from 2 embryos from D14 living embryos was used as one sample for RNA extraction. In total, 100 IVF D8 and 10 IVF D14 blastocysts were prepared for this analysis.

Five RNA samples per group were adjusted to a concentration of 200 pg/μL and prepared for cDNA synthesis using the SMARTer Ultra Low Input RNA Kit for Sequencing-v3 (Takara Bio). The amplified cDNA was fragmented into 200 bp fragments by sonication and processed using the Low Input Library Prep Kit (Takara Bio). After assessing the quality of the constructed libraries, clusters were formed on HiSeq SR FlowCellv3 using cBot (Illumina). Sequencing was performed using a HiSeq 2500 (Illumina) by measuring 50 bp at a time from one direction.

3. Bioinformatics analysis of RNA-seq Before mapping, fluorescence signal correction and data quality confirmation based on quality values were performed, and adapter sequences were removed. Sequence reads were mapped to the bovine reference genome (ensembl UMD3.1). Reads per kilobase of exons per million mapped sequence reads (RPKM) values were calculated and used for expression analysis. Principal component analysis was performed by singular value decomposition of central scale RPKM values. Transcripts with RPKM values greater than 0 were considered expressed. Samples were hierarchically clustered using the group average method and the 1-Pearson correlation coefficient as the distance measure. Differential expression analysis was performed on transcripts whose expression was confirmed using edgeR, an R package. Transcripts with Fisher's exact test p-value less than 0.01 were considered differentially expressed between samples.

4. Immunostaining and confocal microscopy Primary antibodies include goat anti-OCT4 antibody (dilution ratio 1:400, ab27985; Abcam), rabbit anti-CDX2 antibody (dilution ratio 1:200, ab76541; Abcam), rabbit anti-SOX2 antibody (dilution ratio 1:1500, ab92494; Abcam), goat anti-SOX17 antibody (dilution ratio 1:1500, AF1924; R&D Systems), and rabbit anti-NANOG antibody (dilution ratio 1:10000, 500-P236; PeproTech). As secondary antibodies, Alexa Fluor555 goat anti-rabbit IgG polyclonal antibody (ab150082; Thermo Fisher Scientific Inc.) and Alexa Fluor488 donkey anti-goat IgG H&L (ab150129: Abcam) were used.

The zona pellucida of blastocyst stage embryos was removed using 0.05% (w/v) pronase (Sigma-Aldrich). Embryos were fixed for 60 min in phosphate-buffered saline (PBS) containing 4% (w/v) paraformaldehyde (PFA; Wako Pure Chemical Industries) and then in PBS containing 0.2% (v/v) TritonX-100. Permeabilized for 60 minutes. Blocking was then performed for 45 min using BlockingOne (1:5; Nacalai Tesque, Inc.) (blocking buffer) diluted in PBS containing 0.01% (v/v) Tween20. The temperature and incubation time during the primary antibody reaction were changed depending on the target protein; specifically, OCT4, SOX2, SOX17, and NANOG were incubated at 4°C overnight, and CDX2 was incubated at 37°C overnight. Embryos were then washed five times (10 min each) with PBS containing 0.1% (v/v) Triton X-100 and 0.3% (w/v) bovine serum albumin (Sigma-Aldrich), followed by blocking buffer. Incubated with diluted respective secondary antibodies for 30 minutes at room temperature. Nuclei were counterstained with 25 mg/mL Hoechst33342 (Sigma-Aldrich) prepared in PBS containing 0.2% (w/v) polyvinyl alcohol. Fluorescent signals were visualized using a TCS SP5II confocal laser scanning microscope (Leica). The number of specific protein-positive cells was determined by manual counting of blastomeres based on fluorescence images, considering each embryo as one sample. For each cell count, embryos were collected from 2-5 separate experiments.

5. In vitro culture of bovine embryos after the blastocyst stage (on-gel culture)
Blastocyst-stage embryo culture in agarose gel (on-gel culture) was based on previous work focusing on organ culture of testicular tissue. Specifically, agarose powder (Agarose S, Dojindo Laboratories) was diluted with double distilled water to a concentration of 1.5% (w/v) and autoclaved. After pouring the gel into a 10 cm dish and allowing it to solidify at room temperature, gel pieces measuring 10 mm × 10 mm × 5 mm were cut out. The cut out gel was placed in a 4-well dish (Thermo Fisher Scientific Inc.). Then, 30% (v/v) knockout serum replacement (KSR, Thermo Fisher Scientific Inc.), 1× non-essential amino acids (Sigma Aldrich Japan), 1× essential amino acids (Sigma-Aldrich), and 1× insulin-transferrin - 0.5 mL of medium (RPMI1640, Fuji Film Wako Pure Chemical Industries, Ltd.) supplemented with selenium solution (Thermo Fisher Scientific Inc.) was added to each well and incubated overnight at 38.5°C. The used medium was then replaced with the same fresh medium and the gel was immersed to about half its height. IVF D8 blastocyst stage embryos were washed several times with medium and placed on the surface of gel pieces with a small amount of medium using a glass pipette (Figure 2). Five to six blastocyst-stage embryos were placed on individual gel pieces, and subsequent culture was performed at 38.5°C under saturated steam with 5% (v/v) _CO2 . Immediately after the start of culture, the blastocyst-stage embryos were surrounded by the medium introduced during placement. However, once this extra medium was absorbed into the gel, the blastocyst-stage embryos were now exposed to both gas and liquid phases. Light micrographs of embryos were obtained using a DMi8 microscope (Leica).

6. Survival analysis The survival rates of embryos in different conditions were expressed as survival curves using the Kaplan-Meier method and analyzed for significant differences between groups using the log-rank test. Embryonic death was determined based on morphological appearance examined using a stereomicroscope. Three independent experiments were performed under each condition, with 5-6 embryos tested for each condition.

7. Embryo transfer Embryo transfer to the surrogate mother was performed according to previously reported information. Specifically, a progesterone-releasing device (CIDR; InterAg) is placed in the vagina of recipient Holstein cows at Hokkaido University for 7 days before artificial insemination to prevent the start of new follicular development, and to prevent the onset of new follicle development. At the time of extraction, 2 mg of estradiol benzoate and 25 mg of dinoprost were administered, respectively. D10 on-gel embryos were transferred into the ovulated uterine horn of synchronized recipients at D9 after ovulation. A total of 7 D10 embryos were transferred to 5 separate recipients. In the first two transfers, two embryos were transferred into each recipient's uterine horn on the ovulated side. The CIDR was reinserted on the day of transplantation and removed 2 weeks later. Pregnancy was diagnosed by ultrasound (5 MHz, HS101V, Honda Electronics).

8. Quantitative PCR (qPCR)
Total RNA extraction, cDNA synthesis, and subsequent PCR analysis were performed as previously reported. The TE part of the embryo was mechanically isolated under a stereomicroscope. TE isolated from 20 blastocyst-stage embryos, 5 D10 on-gel cultured embryos, and 2 D14 on-gel cultured embryos were pooled and extracted using the ReliaPrep RNA Cell Extraction Kit (Promega). , RNA was extracted according to the manufacturer's protocol.
For gene expression comparison between D10 on-gel cultured embryos and in vivo-derived embryos (Fig. 4G), the TE region was extracted from one embryo for each sample. Ten days after artificial insemination at the Aomori Prefectural Industrial Technology Center Livestock Research Institute, in vivo-derived embryos were collected by non-surgical uterine perfusion.

Samples were collected in triplicate experiments, lysed in lysis buffer, and stored at −80°C until cDNA synthesis. cDNA was synthesized using ReverTra Ace qPCR master mix (Toyobo). qPCR was performed after preparing a reaction mixture using THUNDERBIRD SYBR qPCR Mix (Toyobo). Primer sets used for qPCR analysis are shown in Table 1.

Thermal cycling conditions were 1 cycle of 30 seconds at 95°C (denaturation), followed by 50 cycles of 10 seconds at 95°C (denaturation), 15 seconds at the annealing temperature corresponding to each primer set (primer annealing), and 72°C. It was 30 seconds (extended). Relative mRNA abundance was calculated using YWHAZ (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activating protein zeta), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), and ACTB (actin Calculated by the ΔΔCt method using the geometric mean of β). Results were expressed as the average of 3 to 6 biological replicates for each group in a single qPCR reaction.

9. On-gel culture under different _O2 concentrations
On-gel culture Regardless of the _O2 concentration in the gel, embryo culture up to the blastocyst stage was performed under 5% _O2 .
On-gel culture was performed under 21% O ₂ and 5% CO ₂ (21% O ₂ group) or 5% O ₂ and 5% CO ₂ (5% O ₂ group), and biological The effects of different O ₂ concentrations on the properties were investigated.

10. ROCK inhibitor treatment
100 μM Y27632 (ENZO Life Sciences) or equivalent amount of DMSO was added to the medium during on-gel culture to inhibit ROCK signaling.

11. Self-organizing map (SOM)
SOMs of transcription factors were shown using the R package Kohonen. Genes annotated with the term “transcription factor” in the Gene Ontology (GO) database were considered transcription factors in this analysis.

12. GO term analysis
Functional annotation of transcripts using GO was performed by Ingenuity Pathway Analysis (Qiagen). Two different statistical analyses, Gene Set Enrichment Analysis (GSEA) and Hypergeometric Distribution Test (HyperG) were performed to identify GO terms enriched in differentially expressed genes between D8 and D14 samples. did. For GSEA, expression values for each transcript were calculated as described above based on the change in expression between D8 and D14 samples. For each set of genes annotated with the same GO term, the original test statistic is the expression value of the upregulated genes (transcripts expressed 3 times more in D14 samples than in D8 samples, p < 0.01) divided by the square root of the size of the set. The same procedure was performed on the same number of randomly sampled genes from the upregulated gene set, regardless of GO term annotation, and the resulting values were compared with the original test statistic. This comparison was performed for all combinations, and the upper tail was defined as the quotient of the number of combinations with a test statistic that is the total number of combinations greater than the original test statistic. Upper tails less than 0.01 were considered statistically significant.

In HyperG, we first calculated the frequency of each GO term separately in two groups: all transcripts whose expression was confirmed and up-regulated genes. A GO term that has a higher frequency of occurrence in the upregulated gene group than in the total transcript group, assuming the two groups are of the same size, considers the upregulated gene to be enriched. did. HyperG analysis was performed as previously reported.

13. Preparation of semi-ultrathin and ultrathin sections for light and transmission electron microscopy Embryos were fixed for 2 h at 4 °C using 0.1 M phosphate buffer (PB) containing 2.5% glutaraldehyde (pH 7.4). . After washing three times with PB, the samples were further fixed using 0.1 M PB containing 1% (w/v) OsO ₄ at 4° C. for 2 hours and washed again three times with PB. Samples were dehydrated by immersion in a graded series of ethanols. Ethanol was removed using methyloxirane, and samples were embedded in Epon solution and serially sectioned to form semithin sections (1 μm thick). These sections were then stained with 0.3% basic toluidine blue and observed using a light microscope (DMi8, Leica). Epon-embedded samples were sliced to 80 nm thickness and observed for transmission electron microscopy imaging using JEM-2100 (JEOL Ltd.).

14. statistical analysis
Data from qPCR analysis were expressed as mean ± standard error of the mean (SEM). Significant differences in the data regarding the relative gene expression levels of D8, D10, and D14 TE samples (Fig. 4F) are a result of comparing D8 and D10 and D8 and D14 embryo samples. Data were statistically analyzed using Dunnett's test and R software was used for analysis. A p value less than 0.05 was considered statistically significant.

(result)
1. Development of embryos beyond the blastocyst stage using on-gel culture Unlike mouse and human embryos, reliable culture methods for maintaining post-blastocyst development of bovine embryos have not yet been established. Therefore, to assess the precise time course of lineage segregation after the blastocyst stage, we developed an on-gel culture (Fig. 2A). Specifically, blastocyst-stage embryos were cultured on rectangular agarose gels filled with optimized medium and exposed to both gas and liquid phases during the culture period (Fig. 2A and above). (See "5. In vitro culture of bovine embryos after the blastocyst stage (on-gel culture)"). On-gel cultured blastocyst-stage embryos showed an improved probability of survival beyond the blastocyst stage and showed remarkable similarities with their in vivo counterparts in terms of macroscopic morphology and cell proliferation capacity. (Figure omitted).

Additionally, an embryo transfer test was used to confirm the developmental integrity of the on-gel cultured embryos. In the first two of five transfer trials, two embryos were transferred into the luteal uterine horn of each recipient. In these transfers, one of the recipients was pregnant with a conceptus derived from one embryo. The embryo reached at least day 42 of gestation according to echocardiography, but was unable to complete full-term development. Meanwhile, the other three transfers involved one embryo per recipient, and in two of them pregnancy was confirmed by ultrasound. These transplanted embryos were still alive on August 17, 2021, day 241 of pregnancy (Figure 2B; in Figure 2B, the arrow indicates a fetus with a heartbeat).

Furthermore, the formation of the blastoderm layer was confirmed by both section imaging and qPCR of blastoderm markers (n=2 embryos for section imaging; Figure 2C; arrows indicate the trophoblast layer; arrowheads indicate the embryonic Showing the Hypoblast layer). Due to the delayed development of on-gel cultured embryos compared to in vivo-derived embryos, the subblast layer was clearly visible in in vivo-derived embryos, but was hardly observed in on-gel cultured embryos at D12. D14 embryos established a distinct hypoblast lining the entire trophoblast layer, highlighting the effectiveness of the on-gel culture system. The number of Epi cells (which are more sensitive to oxidative stress than other embryonic cells) in on-gel cultured embryos decreased after D10 (figure omitted).

In order to prevent a decrease in the number of Epi cells during long-term culture, on-gel culture was performed at low oxygen partial pressure (5% O ₂ ) rather than normoxic partial pressure (equivalent to air: 21% O ₂ ). The survival probability, total cell number at D12, and even the number of SOX2-positive cells at D12 were significantly enhanced in embryos cultured under hypoxic conditions compared to embryos cultured under normoxic conditions ( Figure 2D).
Similarly, on-gel culture under hypoxic conditions showed that embryos showed a high degree of similarity to their in vivo counterparts in terms of marker protein localization patterns (n=3 embryos in each condition; Figure 2D). .

2. Separation into three cell lines in a bovine blastocyst stage embryo Next, the separation of cell lines after the blastocyst stage was investigated using on-gel cultured embryos. Immunostaining revealed that OCT4 expression was rapidly downregulated in TE cells from D8.5 (n=6, 9, and 10 for D8.5, D9.0, and D9.5, respectively). Embryos; Figures 3A and 3B).
OCT4 expression in TE cells was almost no longer observed at D9.5. These exclusive expression patterns of OCT4 and CDX2 suggested the establishment of an initial cell lineage separation (Figures 3A and 3B).

On the other hand, SOX2 and SOX17 in ICM cells started to show separate expression between blastomeres from D8.5 and became very exclusive at D9, but SOX2-positive cells and SOX17-positive cells each showed random expression within ICM. The spatial arrangement is shown (Fig. 3C). Thereafter, SOX17-positive PrE cells aligned along the blastocoel (Figures 3C to 3E), similar to in vivo-derived embryos, indicating that the spatial separation of Epi and PrE was completed by D10, as previously reported. This was consistent with the results of the study.
These observations of Epi and PrE progenitors therefore provide the first demonstration that second cell lineage segregation in bovine embryos proceeded by a “Salt and Pepper” distribution within the ICM, similar to that in mouse embryos. provided.

We then use the specific inhibitor Y27632 to modulate the separation process of Epi and PrE in bovine embryos by reducing Rho-associated kinase (ROCK) activity that regulates ICM morphology in mouse blastocyst-stage embryos. The mechanism was investigated.
In controls treated with DMSO, SOX2-positive Epi cells and SOX17-positive PrE cells were clustered within the ICM region (n = 3 embryos daily; Fig. 3F). In contrast, SOX2- and SOX17-positive cells in Y27632-treated embryos began to deviate from the ICM region at D9.0 and then spread throughout the embryo at D10 (n=5 embryos each day; Fig. 3F). On the other hand, the enhanced expression of SOX2 and SOX17 continued under Y27632 treatment, resulting in mutually exclusive expression of SOX2 and SOX17 at D10 (Fig. 3F).

These results showed that inhibition of ROCK signaling disrupted the spatial distribution of ICM cells within the embryo but did not affect the fate restriction of ICM cells to Epi and PrE lineages.

3. Maturation of the bovine trophoblast occurs during the separation of the first and second cell lineages, with distinct morphological features such as microvilli development, appearance of binucleated cells, and gap junction formation. Recognized by characteristics. However, the changes in the transcriptome associated with trophoblast maturation have not yet been elucidated, hindering a fundamental understanding of bovine embryonic development beyond the blastocyst stage.

The expression levels of representative pluripotency markers were significantly downregulated in D14 TE cells (Fig. 4A). SOMs created based solely on transcription factor expression suggested that D14 TEs had acquired a completely different transcriptional network than D8 TEs (Fig. 4B). Functional characterization of trophoblast cells was performed using two types of GO analysis, HyperG and GSEA, which detect GO-terms enriched with upregulated genes (Figure 4C). HyperG calculated test statistics based on the number of genes included in the list, whereas GSEA calculated test statistics based on the expression level value of each gene (Figure 4C). The only GO-term judged to be significant in both analyzes was "extracellular space", indicating enhanced intracellular communication at D14 TE (Fig. 4C).

Based on these results, we investigated changes in ligand and receptor gene expression that accompany interactions in the extracellular space. In particular, we focused on the Wnt, FGF, and VEGF signaling pathways because they are involved in mammalian preimplantation and early pregnancy development.
A heat map showing the changes in RPKM showed that among Wnt genes, Wnt11 was significantly upregulated at D14 TE (Fig. 4D). As seen in the high expression of Wnt5b and Wnt6, it was shown that "non-classical" Wnt signaling may be important for the maintenance of pregnancy in cows, similar to that in mice (FIG. 4D). Upregulation of receptor genes including FGFR1, FGFR2, and VEGFR1 suggested a role for FGF and VEGF signaling in mature trophoblast cells (Fig. 4D).

TEs derived from on-gel cultured embryos may be a preferred model for investigating trophoblast maturation in vitro due to the expression of distinct maturational characteristics such as gap junction formation and binucleated cells (Fig. 4E; in FIG. 4E, the arrow indicates the nuclear membrane and the arrowhead indicates condensed chromatin). Indeed, as shown by RNA-seq, the expression of several upregulated genes during TE maturation was induced at D10 only when embryos were cultured on-gel and using droplet culture. was not induced (figure omitted).

Additionally, qPCR analysis of TEs derived from on-gel cultured embryos revealed that the expression of pluripotency markers OCT4 and SOX2 in TEs was dramatically down-regulated at D10, indicating that pluripotency in TEs was dramatically downregulated at D10. It was suggested that it was virtually lost at the time point (Fig. 4F). This is consistent with the immunofluorescence observation in Figure 3A. Expression of genes predicted to endow cells with properties of mature TEs (GJB1 for gap junction formation, MYL3 for myosin crossbridge, SSLP1 for binucleate formation, and ASCL2 for extensive cell proliferation) was detected from D10. and was dramatically upregulated in D14 on-gel cultured embryos (Fig. 4F). The transcription factors ETS2 and ID2 are involved in the post-implantation development of mouse TE and were upregulated in on-gel cultured embryos at D14 (Fig. 4F). Remarkably, IFNT, one of the downstream targets of ETS2, a ruminant-specific maternal recognition factor, was upregulated in D14 embryos (Fig. 4F).

Finally, the expression of key genes was compared between D10 on-gel cultured embryos and their in vivo-derived counterparts to determine whether on-gel culture mimics physiological conditions with respect to gene expression levels. (Fig. 4G).
Among the pluripotency factors, the expression of OCT4 and NANOG was comparable between groups, whereas the expression of SOX2 was significantly lower in on-gel embryos (Fig. 4G). CDX2, TEAD4, GATA3, ETS2, and ID2, which are important TE-related transcription factors, were downregulated in on-gel cultured embryos compared with in vivo-derived embryos, but not in on-gel cultured embryos. Expression of genes supporting functional maturation of TEs, including IFNT, ASCL2, SSLP1, and CCN2, was comparable to or relatively higher than in in vivo-derived embryos (Fig. 4G).

(Consideration)
Bovine blastocyst stage embryos require more time to establish pregnancy than mouse blastocyst stage embryos. This could lead to differences in the time period required for TEs to acquire unique properties between bovine and mouse embryos. However, the establishment of TE cells, that is, changes in cell characteristics for TE maturation, have not been discussed in detail so far. Furthermore, the temporal continuity of first lineage segregation followed by second lineage segregation has not been investigated in bovine preimplantation embryos.

First, we discovered dramatic transcriptomic changes from D8.0 to D14. This indicates a global property change of TE at the molecular level during this period (figure omitted).
OCT4 is the most important element in maintaining the ICM-specific pluripotency network, as it prevents the functional expression of key TE determinants such as CDX2. Therefore, the expression status of OCT4 can be used as an excellent indicator of TE maturation. Immunostaining analysis revealed that more than 80% of CDX2-positive TE cells expressed OCT4 even at D8.0 (figure omitted). Furthermore, previous studies have shown that OCT4 mRNA expression is indistinguishable between ICM and TE, indicating that first lineage segregation is incomplete in bovine blastocyst stage embryos around D8.0. was. The results of immunostaining performed to analyze the separation of Epi and PrE in ICM revealed that the expression of the Epi marker SOX2 and the Pre marker SOX17 overlap in many ICMs (figures omitted).
This result supports previous studies that identified separation of Epi and PrE using other markers, namely NANOG, an Epi marker, and GATA6, a PrE marker, which showed separate localization in the ICM on D8. The results of the study were slightly different.

SOX17 is one of the late PrE markers in mouse embryos and begins to be expressed in GATA6-positive PrE cells in 64-cell stage embryos. In contrast, bovine SOX17 protein is expressed in almost all blastomeres during the early stages of the blastocyst stage embryo and gradually becomes restricted to the ICM region as the embryo progresses. These differences in marker protein expression patterns may reflect species-specific developmental plasticity. One striking example of this is that, unlike in mice, bovine ICMs mechanically isolated from whole embryos can survive from development to term.

On-gel culture methods were developed to overcome the developmental limitations of traditional methods such as droplet-based cultures and allow the detailed time course of lineage segregation beyond the blastocyst stage to be determined. do.

Loss of OCT4 expression from TE cells was demonstrated to be complete at D9.5, supporting complete loss of TE cell pluripotency (FIGS. 3A and 3B).
This result was consistent with the results of previous studies showing that OCT4 expression in TE cells was dramatically reduced at D9 and virtually undetectable at D11 in vivo. Interestingly, we found that the segregation of a second cell lineage in bovine embryos proceeds in parallel to the first. By D9.5, SOX2 and SOX17 expression became exclusive between blastomeres, after which PrE cells aligned along the blastocoel, at which point Epi and PrE were also spatially separated (Fig. 3C). This phenomenon is quite different from mouse embryos, where a second cell separation occurs after the completion of the first cell separation, and may be a mechanism of early embryonic development unique to ruminants.

The second segregation in bovine embryos is a partially cell-autonomous process, as SOX2 and SOX17 now show exclusive expression even when ICM morphology is altered by Y27632 treatment. was shown (Fig. 3F).
Y27632 treatment before blastocyst dorsal formation was shown to increase the number of CDX2-positive TE cells on D7.5, indicating that ROCK signaling contributes to cell lineage determination in bovine blastocysts. suggested.

In this example, we show that dramatic down-regulation of pluripotency markers, changes in transcriptional networks, and functional analysis using GO-terms as determinants affect bovine trophoblast maturation during and after two lines of lineage segregation. Persistence was demonstrated (Figures 4A-4C).

FGF signaling may contribute to successful pregnancy. This is supported by previous studies in which conceptuses from heifers with high fertility showed higher FGFR2 expression than conceptuses from heifers with low fertility. ing.
Therefore, the high expression of FGFR shown in this example may be involved not only in the proper differentiation of trophoblasts but also in the crosstalk with the maternal endometrium (Fig. 4D).

The Wnt signaling pathway has been suggested to contribute to the development of bovine preimplantation embryos, and addition of DKK1, an activator of the non-classical WNT pathway, increases pregnancy rates after embryo transfer.
The upregulation of Wnt11 in D14 TE samples (Fig. 4D) supported the possibility that the non-classical Wnt pathway plays an important role in bovine preimplantation embryonic development.

Furthermore, time-dependent changes in marker genes were examined using on-gel cultured embryos (Fig. 4F). Elevated expression levels of these genes indicated that D10 embryos were in the preparatory stage of exhibiting morphological changes characteristic of mature TE. The appearance of binucleate cells was gradually detected only after D16 in vivo.
On the other hand, the expression of the pregnancy recognition factor IFNT, its upstream regulatory factor ETS2, and the transcription factor ID2, which is predominantly expressed in TE, was found to be increased in D12 on-gel cultured embryos. It was suggested that the derived mature TE cells were physiologically functional at D14 (Fig. 4F).

A series of results demonstrated for the first time that bovine TE cells begin functional maturation during two lineage segregations. We also observed that the expression status of several important transcription factors was different between on-gel cultured samples and in vivo-derived D10TE samples (Fig. 4G). The intrauterine environment has been shown to influence embryonic gene expression of factors such as OCT4, SOX2, and CDX2. In this example, long-term in vitro culture may have impaired the proper establishment of this transcriptional network.
Future studies plan to define the critical factors required to initiate the correct pattern of gene expression in preimplantation embryos by manipulating the environment surrounding the embryo in on-gel culture. There is.

It is also important to note that the expression of genes promoting mature TE function was comparable or somewhat greater in on-gel cultured embryos than in in vivo-derived embryos. Some of these increases in gene expression related to TE function were only observed when embryos were cultured on-gel, but not in droplet-cultured embryos (Figure, omitted).

In addition to successful implantation of on-gel cultured embryos and continued fetal development in utero (Figures 2A and 2B), on-gel culture methods have been shown to maintain developmental integrity at least until D10. It was done.

In conclusion, this example demonstrated how immature bovine blastocyst stage embryos lose their embryological plasticity and establish different cell lineages. Separation of the first and second cell lineages ends simultaneously at D9.5, which is accompanied by a rapid qualitative change of TE cells into mature trophoblasts.
The results obtained from this example provided deeper insight into the implantation strategies employed in non-rodent mammals, including humans.

[Example 2]
Next, using the on-gel culture method established in Example 1, the survival rate of the on-gel cultured embryos was verified.

(Raw materials and methods)
All experiments were approved and performed by the Hokkaido University Regulatory Committee for the Care and Use of Laboratory Animals.

1. Embryo transfer of D10 on-gel cultured embryos In Example 1, five embryo transfer sessions were performed using D10 on-gel cultured mature blastocyst stage embryos. Recipient Holstein heifers at Hokkaido University were monitored for ovulatory cycles by an ultrasound machine (HS-2100V; Honda Electronics) with a linear probe (10 MHz, HLV-475; Honda Electronics) prior to synchronization of ovulation. The normality of the uterine contents was examined repeatedly.
Endometritis was treated with intrauterine injections of antibiotics when necessary. Heifers were treated with 2 mg benzoic acid for 7 days (day −10 to day −2, IVF day designated as day 0) using an intravaginal drug release device (CIDR1900; Zoetis Japan). Estradiol (E2) (Ovahormon injection, ASKA Animal Health Co., Ltd) and 25 mg of dinoprost (prostaglandin F _2α ; PGF _2α ) (Pronalgon F, Zoetis Japan) were administered at the time of device insertion and removal, respectively. and synchronized ovulation.

On day 0 (the day of ovulation), recipients received 100 μg of gonadotropin-releasing hormone GnRH. Ovaries were examined daily by ultrasound (5 MHz, HS101V, Honda Electronics), and ovulation was confirmed in one heifer on day 1 and in the other on day 2.

Embryo transfer (ET) was performed in the morning of day 10. Table 2 shows details of the experimental schedule.

The CIDR device was inserted for 2 weeks starting on day 6 to facilitate conception. The presence and location of the corpus luteum was confirmed by ultrasonography on the 8th day. Embryo transfer was performed on the 10th day. Embryos and medium were transferred to plastic straws (005592, IMV Technologies) and loaded immediately. It was placed in a disposable ET gun (MO5, Misawa Medical Industry Co., Ltd.) and quickly transplanted to the uterine horn ipsilateral to the corpus luteum. At the time of embryo transfer, caudal epidural anesthesia with lidocaine hydrochloride (Xylocaine (registered trademark) 2v/v%, Sandoz Co., Ltd.) was performed. Pregnancy was diagnosed by ultrasound examination (HS101V) 30 days after pregnancy. Two recipients with visible gestational sacs carried pregnancies to term.

2. Preparation of bovine embryos by conventional culture methods in microdroplets Recovery of bovine oocytes, IVM, IVF, and subsequent in vitro culture (IVC) to the blastocyst stage were performed as previously described and as previously described. Specifically, cumulus-oocyte complexes (COCs) collected from slaughterhouse-derived ovaries were cultured in TCM-199 medium (Thermo Fisher Scientific Inc.) at 38.5 °C and saturated with 5% _CO2 in air. Cultured under water vapor for 20-22 hours. Eggs matured to second meiotic metaphase were transferred to Brackett and Oliphant (BO) medium containing 2.5 mM theophylline (Fuji Film Wako Pure Chemical Industries, Ltd.) and 7.5 μg/mL heparin sodium salt (Nacalai Tesque Co., Ltd.). . Subsequently, the frozen and thawed semen was washed in BO medium by centrifugation at 600 × g for 7 minutes. Sperm were added to the COC at a final concentration of 5×10 ⁶ cells/mL. Presumably fertilized eggs were incubated at 38.5 mL under saturated water vapor of 5% (v/v) CO ₂ and 5% (v/v) O ₂ using mSOFai medium, removing cumulus cells by pipetting after 12 h incubation. Cultured at ℃ for 8 days.

3. On-gel culture of bovine immature blastocyst stage embryo Agarose powder (Agarose S, Dojindo Laboratories) was diluted to a concentration of 1.5% (w/v) with double-distilled water and autoclaved. After pouring the gel into a 10 cm dish and allowing it to solidify at room temperature, gel pieces measuring 10 mm × 10 mm × 5 mm were cut out. The cut out gel was placed in a 4-well dish (Thermo Fisher Scientific Inc.). Then, 30% (v/v) knockout serum replacement (KSR, Thermo Fisher Scientific Inc.), 1× non-essential amino acids (Sigma Aldrich Japan), 1× essential amino acids (Sigma-Aldrich), and 1× insulin-transferrin - 0.5 mL of medium (RPMI1640, Fuji Film Wako Pure Chemical Industries, Ltd.) supplemented with selenium solution (Thermo Fisher Scientific Inc.) was added to each well and incubated overnight at 38.5°C. The used medium was then replaced with the same fresh medium and the gel was immersed to about half its height. IVF D8 blastocyst stage embryos were washed several times with medium and placed on the surface of a gel piece with a small amount of medium using a glass pipette. Five to six blastocyst-stage embryos were placed on individual gel pieces, and subsequent culture was performed at 38.5 °C under saturated steam with 5% (v/v) CO _and 5% (v/v) _O. Ta. Immediately after the start of culture, the blastocyst stage embryos were surrounded by the medium introduced during placement. However, once this extra medium was absorbed into the gel, the blastocyst-stage embryos were now exposed to both gas and liquid phases.

4. Blood Sampling and Blood Tests On days 1, 3, 7, 14, 21, and 28 postpartum, calves were placed in the jugular vein using a sodium heparin tube (Venoject II; Terumo) for analysis of biochemical components. Blood samples were collected from. After blood collection, samples were immediately centrifuged at 1,660 × g for 15 min at room temperature to separate plasma and stored at −30°C until analysis. Concentrations of plasma glucose, blood urea nitrogen, total cholesterol, total protein, albumin, calcium, inorganic phosphorus, γ-glutamyl transferase, and ammonia were measured using a DRI-CHEM 3000V clinical hematology analyzer (Fujifilm). .

(result)
In order to verify the survival rate of on-gel cultured embryos, ET was performed according to the schedule shown in Table 2 above.

Of the five ETs performed, two recipient Holstein heifers were confirmed to be pregnant, but full development of the corresponding conceptuses could not be confirmed.

Furthermore, considering the possibility that IVC is a contributing factor to the development of large offspring syndrome (LOS), we evaluated the physical condition of newborn calves derived from on-gel cultured embryos. LOS is characterized by large birth size and placental abnormalities. These are the major issues of concern regarding the production of cattle from in vitro produced (IVP) embryos. Since on-gel culture is an IVC system that involves long-term culture, for practical application it is necessary to evaluate the integrity of fetuses and placentas derived from on-gel cultured embryos.

In this example, full-term development of two on-gel cultured embryos after ET was confirmed, their birth weights were measured, and blood biochemical analysis was performed. In addition, postnatal body weight was monitored and postnatal blastoderm was determined. The results showed that in on-gel culture, the preimplantation development seen in the in vivo counterpart was recapitulated at D10.

These findings thus demonstrated that this culture system can be used to generate developmentally mature blastocysts for animal production and bovine developmental biology studies.

Calves were born vaginally without assistance from each of the two pregnant recipients with D10 on-gel cultured embryos on September 21 and November 29, 2021, respectively (Figure 5 top left and top right). All were breathing spontaneously and appeared normal.
Birth weights (40 kg and 46 kg) and gestational age (286 and 285 days) were within typical ranges, and the animals were alive and free of disability as of March 25, 2022.
Therefore, we confirmed the full-term development of D10 on-gel cultured embryos via ET and demonstrated the reproducibility of cattle production using mature blastocyst stage embryos produced by the on-gel culture system. .

The postpartum weights of the calves delivered by the recipients were 2.6 kg and 3.0 kg, respectively, but no evidence of morphological abnormalities was observed (lower left of Figure 5). The number of postnatal blastoderms was counted by manually separating each blastoderm from birth (162 and 96). Both postnatal weight and blastoderm number were within their normal ranges.
Therefore, these results indicated that the TE of mature blastocysts cultured on-gel had the ability to form a placenta that supported proper fetal development to term.

To further confirm that newborn calves derived from mature blastocysts obtained by on-gel culture are healthy, calves derived from both on-gel culture and control artificial insemination (AI) were collected. The metabolic profile was investigated based on the analysis of peripheral blood samples.
From the analysis of nine biochemical indicators (plasma glucose, blood urea nitrogen, total cholesterol, total protein, albumin, calcium, inorganic phosphorus, γ-glutamyl transferase, and ammonia) that represent important indicators, in each case , concentrations in calves derived from on-gel cultures were found to be comparable to values obtained for calves derived from AI at 0, 3, 7, 14, and 28 days of age (Table 3). .

Among the unresolved issues associated with IVP systems for cattle production, LOS is associated with increased incidence of dystocia and placental retention. Although the factors contributing to abnormal fetal growth have not yet been fully determined, the addition of serum in IVC has been confirmed to reduce post-ET survival and cause abnormal fetal growth, including LOS. There is. Notably, two calves derived from on-gel cultured mature blastocysts showed no abnormal phenotype during the neonatal period, and birth weight, placental weight, blastoderm number, and metabolic profile were It was comparable to AI-derived calves.
Therefore, these results suggested that the on-gel culture system using the serum substitute KSR instead of fetal bovine serum resets the deleterious effects of serum on fetal and placental growth.

This example suggests that on-gel culture can be used to promote the development of embryologically mature blastocyst stage embryos that have undergone two rounds of cell separation. On-gel culture allows obtaining higher cell numbers than traditional droplet culture and biopsy combinations, making this system a new method for genetic evaluation of embryos during the pre-implantation stage. there is a possibility.

Furthermore, the on-gel culture system reproduced levels of cell differentiation comparable to those observed in their in vivo counterpart embryos, at least until D10 after the initiation of IVC.
Therefore, we believe that the on-gel culture system can contribute to a series of analyzes to evaluate preimplantation development unique to bovines. Furthermore, considering that long-term IVC with on-gel culture allows for a more stringent selection of embryos for embryo transfer, on-gel culture systems can be used to increase the efficiency of cows by embryo transfer. It is thought that this can promote production.

According to the in vitro culture method for bovine embryos of the present embodiment, rapid degeneration and loss of differentiation ability of embryos after the blastocyst stage are suppressed, and in vitro cultured embryos have gene expression and morphological characteristics similar to living embryos. is obtained. The bovine embryo of this embodiment has gene expression and morphological characteristics similar to those of a living embryo, with rapid degeneration and loss of differentiation ability of the embryo after the blastocyst stage being suppressed. The cow production method of the present embodiment uses bovine embryos obtained by the above-mentioned in vitro culture method of bovine embryos, and can contribute to the breeding and improvement of desired cows.

1... Gel, 2... Medium, 3... Culture container, 10... Bovine embryo in vitro culture system, B... Bovine blastocyst stage embryo

Claims (9)

A method for culturing bovine embryos in vitro, comprising a culturing step of culturing bovine blastocyst-stage embryos on a gel impregnated with a medium. The in vitro culture method for bovine embryos according to claim 1, wherein in the culturing step, the culture is carried out in the presence of oxygen of 3 v/v % or more and 7 v/v % or less. The method for culturing bovine embryos in vitro according to claim 1 or 2, wherein in the culturing step, the embryos are cultured for one day or more. The in vitro culture method for bovine embryos according to claim 1 or 2, wherein the medium contains a serum substitute in an amount of 10 v/v % or more and 50 v/v % or less based on the volume of the medium. The method for culturing bovine embryos in vitro according to claim 1 or 2, wherein the gel is an agarose gel. A bovine embryo obtained by the in vitro culture method for bovine embryos according to claim 1 or 2. A bovine embryo obtained by in vitro fertilization,
A bovine embryo in which one or more genes selected from the group consisting of IFNT, GJB1, MYL3, ETS2, ID2, SSLP1, and ASCL2 are expressed 10 days after in vitro fertilization. 10 days after in vitro fertilization, the expression level of one or more genes selected from the group consisting of OCT4, NANOG, and SOX2 is 1/10 or less of the expression level of the same gene 8 days after in vitro fertilization. 7. The bovine embryo described in 7. A method for producing cattle, comprising a transplanting step of transplanting a bovine embryo obtained by the in vitro culture method for bovine embryos according to claim 1 or 2 into an embryonic cow to impregnate it.

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