Composition for extending telomeres of chromosomes in vitro, method for producing embryo, embryo, and embryo-receiving livestock animal

Abstract

To provide a composition and the like capable of extending the telomeres of chromosomes in an embryo.SOLUTION: Provided is a composition for extending the telomeres of chromosomes in embryos in vitro, the composition comprising TGFβ1 or AGT.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a composition for extending chromosomal telomeres in vitro, a method for producing an embryo, an embryo, and an embryo recipient livestock animal. [Background technology]

[0002] Embryo transfer is widely used in Japanese cattle production and human reproductive medicine. Telomeres are structures at the ends of chromosomes that play a role in maintaining the integrity of the genome and are shortened, for example, by cell division and oxidative stress associated with aging. Cells with shortened telomeres stop dividing and their functions decline. It is known that in mice, cattle, and other animals, embryonic telomeres are elongated toward the blastocyst stage after fertilization (Non-Patent Documents 1 and 2). In a study in which the telomere length of an embryo was extended using a chimera created from ES cells and the offspring were compared, it was reported in mice that the telomere length of the embryo affects the telomere length of the offspring and also affects the speed of wound recovery (Non-Patent Document 3). It has been reported that when the father is aging, the telomere length of the embryo is shortened, the telomere of the offspring is also shortened, and the lifespan of the offspring is also shortened (Non-Patent Documents 4 and 5). Similarly, it has been reported in mice that the telomeres of eggs are shortened due to maternal aging (Non-Patent Document 6). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Nat Cell Biol. 2007;12:1436-41. [Non-Patent Document 2] Reprod Domest Anim 2015;50:58-67. [Non-Patent Document 3] Nat Commun. 2016;7:11739. [Non-Patent Document 4] Mitochondrion. 2023; 68:105-113. [Non-Patent Document 5] Proc Natl Acad Sci US A. 2018;115:E2348-E2357. [Non-Patent Document 6] Reprod Biol Endocrinol. 2013;11:108. Summary of the Invention [Problem to be solved by the invention]

[0004] Therefore, creating a culture environment that promotes telomere elongation of chromosomes may be able to affect not only the development of fertilized eggs and embryos but also the lifespan and prognosis of the offspring.

[0005] An object of the present invention is to provide a composition and the like capable of extending telomeres of chromosomes in an embryo. [Means for solving the problem]

[0006] The present inventors have conducted extensive research to solve the above problems, and as a result have found that a composition containing TGFβ1 or AGT can elongate telomeres of chromosomes in an embryo, thereby completing the present invention.

[0007] That is, the present invention is as follows. [1] A composition for in vitro elongation of chromosome telomeres in an embryo, comprising TGFβ1 or AGT. [2] The composition described in [1], which contains TGFβ1. [3] The composition described in [1] or [2], which contains AGT. [4] The composition according to any one of [1] to [3], for culturing a fertilized egg or embryo within 48 hours after fertilization. [5] The composition according to any one of [1] to [4], wherein the composition contains follicular fluid. [6] The composition according to any one of [1] to [5], which increases the cell number of a blastocyst stage embryo during the development of the embryo. [7] The composition according to any one of [1] to [6], which promotes development of the embryo. [8] The composition according to any one of [1] to [7], wherein the embryo is a bovine embryo. [9] [1] to [8], comprising culturing a fertilized egg or embryo in vitro in the presence of the composition. A method for producing embryos with extended chromosomal telomeres.

[10] [9] An embryo produced by the method described in [9].

[11]

[10] A recipient livestock animal having an embryo according to the above. Effect of the Invention

[0008] According to the present invention, it is possible to provide a composition and the like capable of extending telomeres of chromosomes in an embryo. [Brief description of the drawings]

[0009] [Figure 1] Telomere length per blastomere in blastocysts after addition of TGFβ1. The control group is set at 1. * indicates a significant difference. P<0.05 [Diagram 2] Telomere length per blastomere in blastocysts after AGT addition. The control group is set at 1. * indicates a significant difference. P<0.05 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Below, we will explain in detail the form for implementing the present invention (hereinafter referred to as the "present embodiment"). However, the present invention is not limited to this, and various modifications are possible without departing from the gist of the present invention.

[0011] The present inventors have found that a composition comprising TGFβ1 or AGT is capable of lengthening telomeres of chromosomes in embryos in vitro. Thus, one aspect of the present invention relates to a composition for in vitro elongation of chromosomal telomeres in an embryo. One aspect of the invention relates to a method for extending telomeres of chromosomes in an embryo. One aspect of the present invention relates to a method for producing an embryo having elongated chromosomal telomeres. One aspect of the present invention relates to an embryo in which the telomeres of the chromosomes have been elongated. One aspect of the present invention is an embryo-recipient livestock animal having an embryo with elongated chromosomal telomeres. One aspect of the invention relates to the use of TGFβ1 or AGT to extend chromosomal telomeres in embryos in vitro. One aspect of the present invention relates to the use of TGFβ1 or AGT for use in the preparation of a composition for the in vitro extension of chromosomal telomeres in an embryo.

[0012] [Composition] The composition of the present embodiment contains TGFβ1 or AGT and is used to extend telomeres of chromosomes in an embryo in vitro. The composition of the present embodiment may be used to produce "Nodai fertilized eggs".

[0013] TGFβ1 is transforming growth factor-β1, a cytokine belonging to the TGF-β family. TGFβ1 may be an active form that binds to a TGF-β receptor, or a latent form in which active TGFβ1 is bound to LTBP (latent TGF-β binding proteins) and LAP (latency-associated peptide), but the active form is preferred. TGFβ1 may be derived from the same animal as the embryo to which the composition of the present embodiment is applied. TGFβ1 may be derived from humans, cows, pigs, rats, mice, rabbits, sheep, cats, dogs, monkeys, or the like. TGFβ1 may have, for example, the amino acid sequence of SEQ ID NO: 1 of human-derived TGFβ1, or may have an amino acid sequence having 80% or more, 85% or more, 90% or more, or 94% or more sequence identity to the amino acid sequence of SEQ ID NO: 1.

[0014] [ka]

[0015] AGT stands for angiotensinogen precursor, and angiotensin I and angiotensin II as the active forms of angiotensinogen. AGT may be derived from the same animal as the embryo to which the composition of the present embodiment is applied. AGT may be derived from humans, cows, pigs, rats, mice, rabbits, sheep, cats, dogs, monkeys, or the like. AGT may be contained in the composition of the present embodiment in any form of angiotensinogen precursor, angiotensin I, and angiotensin II, or as a mixture thereof, but is preferably contained in the form of angiotensin II. The AGT contained in the composition of this embodiment may have, for example, the amino acid sequence of SEQ ID NO: 2 of human angiotensinogen, or may have an amino acid sequence having 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more sequence identity to the amino acid sequence of SEQ ID NO: 2, or may be angiotensin I or angiotensin II as an active form of angiotensinogen precursor having the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more sequence identity to the amino acid sequence of SEQ ID NO: 2. The AGT contained in the composition of this embodiment may be human angiotensin II, or may have an amino acid sequence having 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more sequence identity to the amino acid sequence of human angiotensin II.

[0016] [ka]

[0017] The composition of this embodiment may contain either TGFβ1 or AGT, or may contain both TGFβ1 and AGT. The composition of this embodiment may contain components other than TGFβ1 and AGT, so long as they do not inhibit the effects of the present invention. Such components include components that can be contained in culture fluids such as synthetic oviductal fluid (SOF) that can be used in the culture of fertilized eggs or embryos. Components other than TGFβ1 and AGT may be added in the form of synthetic oviductal fluid.

[0018] Components that can be contained in a culture medium that can be used in culturing a fertilized egg or an embryo include, for example, inorganic salts, lactate, pyruvate, amino acids (e.g., glutamine, glycine, taurine, etc.), glucose, streptomycin, penicillin, glucose, phenol red, etc. Examples of inorganic salts include sodium chloride, potassium chloride, calcium chloride, magnesium chloride, potassium hydrogen phosphate, sodium bicarbonate, etc.

[0019] The composition of this embodiment may contain follicular fluid. The composition of this embodiment may be follicular fluid collected from an animal. The composition of this embodiment may be a composition in which a specific component is added to follicular fluid collected from an animal. The specific component may be TGFβ1 or AGT. The composition of this embodiment may be a composition in which a specific component is removed from follicular fluid collected from an animal. When the composition of the present embodiment contains follicular fluid, the follicular fluid may contain TGFβ1 or AGT, or the composition may contain TGFβ1 or AGT separately from the follicular fluid. The animal from which the follicular fluid is collected is preferably an animal from which a fertilized egg or an embryo is derived, but follicular fluid collected from an animal of a different species may be used.

[0020] The concentration of TGFβ1 in the composition of this embodiment may be, for example, 1 ng / mL or more, and is preferably 3 ng / mL or more, 5 ng / mL or more, 8 ng / mL or more, or 10 ng / mL or more. In the above range, the concentration of TGFβ1 is preferably 50 ng / mL or less, 40 ng / mL or less, 30 ng / mL or less, 25 ng / mL or less, or 20 ng / mL or less.

[0021] The concentration of AGT in the composition of this embodiment may be, for example, 0.1 pg / mL or more, preferably 0.3 pg / mL or more, 0.5 pg / mL or more, 0.8 pg / mL or more, or 1 pg / mL or more. In the above range, the concentration of AGT is preferably 30 pg / mL or less, 20 pg / mL or less, 10 pg / mL or less, 8 pg / mL or less, or 5 pg / mL or less.

[0022] The concentrations of components other than TGFβ1 and AGT in the composition of this embodiment are not particularly limited and may be similar to those of synthetic oviductal fluid or follicular fluid.

[0023] The composition of the present embodiment is a composition for extending telomeres of chromosomes in an embryo in vitro, and is distinguished from a composition for extending telomeres of chromosomes in an embryo in vivo. The composition of the present embodiment may be an artificially prepared composition. The artificially prepared composition also includes a composition produced in vivo to which a specific component has been added, and a composition produced in vivo to which a specific component has been removed.

[0024] The composition of the present embodiment is used to elongate chromosomal telomeres in an embryo in vitro, in other words, the composition of the present embodiment is used to obtain an embryo in which chromosomal telomeres are elongated compared to an embryo obtained without applying the composition of the present embodiment. The composition of the present embodiment is used to produce an embryo in vitro in which chromosomal telomeres are elongated. The embryo may have chromosomal telomeres elongated in a state ranging from an 8-cell stage embryo to a blastocyst stage embryo.

[0025] In the present specification, a fertilized egg refers to a cell in which a sperm has entered the egg and fusion of a male pronucleus and a female pronucleus has been completed. In this embodiment, the fertilized egg may be obtained by in vitro fertilization, and the fertilized egg obtained by in vitro fertilization may be frozen and stored, and then thawed for use.

[0026] As used herein, an embryo refers to a fertilized egg that has begun cell division after fertilization. In the present invention, an embryo may be an embryo up to the blastocyst stage, for example, a cleavage stage embryo, a morula, or a blastocyst stage embryo.

[0027] The composition of the present embodiment extends the telomere of a chromosome in an embryo in vitro, but it is not necessarily required to apply the composition in the state of an embryo. The composition may be applied in the state of a fertilized egg, and the telomere of a chromosome in the resulting embryo may be extended. That is, the composition of the present embodiment may be applied to a fertilized egg or an embryo, and the telomere of a chromosome in the resulting embryo is extended. The composition of the present embodiment may be applied, for example, until the fertilized egg becomes a blastocyst stage embryo, or until the fertilized egg leaves the blastocyst stage embryo. The composition of the present embodiment may be applied, for example, until the fertilized egg becomes an 8-cell stage embryo. The composition of the present embodiment may be applied, for example, to a fertilized egg or an embryo within 48 hours (for example, 1 to 48 hours, 3 to 40 hours, 5 to 30 hours, 9 to 28 hours, or 12 to 25 hours) after fertilization treatment.

[0028] The composition of the present embodiment may be a culture medium for culturing a fertilized egg or an embryo. When a fertilized egg is cultured using the composition of the present embodiment as a culture medium, the telomeres of the chromosomes are elongated in the embryo that develops from the fertilized egg. When an embryo is cultured using the composition of the present embodiment as a culture medium, the telomeres of the chromosomes are elongated in the embryo. The composition of the present embodiment may be a culture medium for culturing a fertilized egg or an embryo until the fertilized egg becomes a blastocyst stage embryo or until the fertilized egg leaves the blastocyst stage embryo. The composition of the present embodiment may be a culture medium for culturing a fertilized egg or an embryo until the fertilized egg becomes an 8-cell stage embryo, for example. The composition of the present embodiment may be used as a culture medium for culturing within 48 hours (for example, 1 hour to 48 hours, 3 hours to 40 hours, 5 hours to 30 hours, 9 hours to 28 hours, or 12 hours to 25 hours) after fertilization treatment.

[0029] The embryos whose telomere length is extended by the composition of the present embodiment may be human embryos or non-human embryos, such as cows, pigs, rats, mice, rabbits, sheep, cats, dogs, monkeys, etc. The embryos may be livestock animals, particularly cows. The telomere length extension by the composition of this embodiment does not include that performed in the human body. In addition, biological components that extend telomeres of chromosomes in an embryo in the body, including TGFβ1 or AGT, are distinguished from the composition of this embodiment.

[0030] The composition of the present embodiment is used to elongate telomeres of chromosomes in an embryo, and may further increase the cell number of a blastocyst stage embryo during the process of embryo development, promote embryo development, and / or increase the copy number of mitochondrial DNA (mtDNA) in the embryo. Sahin et al., Nature 2011;7334;359-365 reports that inhibition of telomere elongation leads to a decrease in mtDNA copy number and mitochondrial dysfunction.

[0031] [method] The method for producing an embryo with elongated chromosomal telomeres of this embodiment (hereinafter also simply referred to as the "method for producing an embryo of this embodiment") comprises culturing a fertilized egg or embryo in vitro in the presence of the composition of this embodiment (hereinafter also simply referred to as the "culture step"). The composition of this embodiment may be in any of the forms described above. The method for producing an embryo of this embodiment can also be called a "Nature University embryo production method", and the embryo obtained thereby may be called a "Nature University fertilized egg".

[0032] The above culture may be culture of a fertilized egg or embryo until the fertilized egg becomes a blastocyst stage embryo or until the fertilized egg leaves the blastocyst stage embryo, and is preferably culture of a fertilized egg or embryo until the fertilized egg becomes an 8-cell stage embryo. The above culture may be culture of a fertilized egg or embryo within 48 hours after fertilization (e.g., 1 to 48 hours, 3 to 40 hours, 5 to 30 hours, 9 to 28 hours, or 12 to 25 hours). The above culture may be started from an unfertilized egg after fertilization, or may be performed by selecting a fertilized egg that has been confirmed to be fertilized. The above culture may be started within 30 hours after fertilization (e.g., 0 to 30 hours, 1 to 28 hours, 5 to 25 hours, 9 to 23 hours, or 12 to 20 hours).

[0033] The culture time in the above culture step may be, for example, until the embryo becomes a blastocyst stage embryo or until the embryo leaves the blastocyst stage, but is preferably until the fertilized egg becomes an 8-cell stage embryo. The culture time may be, for example, from 10 hours to 60 hours, from 15 hours to 50 hours, from 20 hours to 40 hours, or from 25 hours to 35 hours.

[0034] The method for producing an embryo of this embodiment may include another step before or after the above-mentioned culture step. For example, the method for producing an embryo of this embodiment may include a fertilization treatment step of contacting an egg with sperm in vitro prior to the above-mentioned culture step. The fertilization treatment step may be a step of contacting an egg with sperm in vitro to produce a fertilized egg, or a step of producing a fertilized egg by in vitro fertilization using sperm and an egg. The fertilization treatment step may be performed in the presence of the composition of this embodiment, or in the absence of the composition of this embodiment.

[0035] The method for producing an embryo according to the present embodiment may include a selection step of selecting fertilized eggs that have been confirmed to be fertilized after the fertilization treatment step. The selection step may be a step of selecting fertilized eggs by, for example, confirming fertilized eggs using a microscope or the like. The fertilized eggs selected by the selection step may be subjected to the above-mentioned culture step.

[0036] The method for producing an embryo of this embodiment may include a step of further culturing in the absence of the composition of this embodiment after the above-mentioned culturing step. When culturing in vitro, the culture medium in such culture may be a culture medium usually used for embryo development. Alternatively, the embryo may be cultured for a certain period of time in the oviduct of an animal. Such culture may be until the embryo becomes a blastocyst stage embryo, or until the embryo leaves the blastocyst stage embryo.

[0037] The method for extending telomeres of chromosomes in an embryo of the present embodiment includes culturing a fertilized egg or embryo in vitro in the presence of the composition of the present embodiment. The composition of the present embodiment may be in any of the above-mentioned forms. The culturing in such a method may be the same as the culturing step in the method for producing an embryo of the present embodiment.

[0038] [Embryos and livestock recipients] The embryo of this embodiment is produced by the above-mentioned method for producing an embryo of this embodiment, and has elongated chromosomal telomeres. The embryo of this embodiment has elongated telomere length compared to an embryo produced by a method different from the method for producing an embryo of this embodiment. The embryo of this embodiment has elongated telomere length compared to an embryo produced by in vitro fertilization (excluding embryos produced by the method for producing an embryo of this embodiment). The embryo of this embodiment may be in a frozen state. The embryo of this embodiment may be an embryo from the 8-cell stage to the blastocyst stage. The embryo of this embodiment can be said to be an "Agricultural University fertilized egg".

[0039] By implanting the embryo of this embodiment into a recipient animal, an animal derived from the embryo can be produced. The produced animal may have an extended life span and an improved wound healing rate because it is derived from an embryo with elongated telomeres. The recipient animal may be a livestock animal, such as a cow or a pig, or may be a cow.

[0040] The embryo-recipient livestock animal of this embodiment is produced by the above-mentioned method for producing an embryo of this embodiment, and has an embryo with elongated chromosomal telomeres. The embryo-recipient livestock animal of this embodiment can be produced by implanting an embryo produced as described above into an embryo-recipient animal. An animal with elongated telomere length can be produced by the embryo-recipient livestock animal of this embodiment. The implantation of the embryo may be performed when the embryo is at the blastocyst stage. The embryo-recipient livestock animal may be a cow or a pig, and may be a cow. EXAMPLES

[0041] The present invention will be described in more detail below using examples and comparative examples, but the present invention is not limited to the following examples.

[0042] First, we added the telomerase inhibitor TMPyP4 to the developmental medium of in vitro fertilized bovine embryos to create blastocysts with shortened telomeres. As a control, we created blastocysts without adding TMPyP4, which did not shorten telomeres. We subjected these embryos to RNA-seq analysis to examine differentially expressed genes. We investigated pathways related to differentially expressed genes. We also used Ingenuity Pathway Analysis to analyze upstream factors that predict the effects of telomere elongation from differentially expressed genes.

[0043] As a result, the differentially expressed genes were associated with calcium signaling, TGFβ signaling, and ECM-receptor interaction pathways. In addition, TGFβ1 and AGT were presumed to be factors that have the same effect as telomere elongation among multiple growth factors.

[0044] Next, the telomere elongation effect of TGFβ1 and AGT was confirmed. Pronuclear embryos 18 hours after fertilization were cultured for 30 hours in a culture medium containing synthetic oviduct fluid to which TGFβ1 or AGT had been added (the concentrations were as follows: TGFB1: 10 ng / mL, and AGT: 1 pg / mL). The embryos were then cultured for 5 days in synthetic oviduct fluid to which TGFβ1 or AGT had not been added until they reached the blastocyst stage. The composition of the synthetic oviduct fluid was as described in Theriogenology. 1992 May;37(5):963-78. Human-derived TGFβ1 having the amino acid sequence of SEQ ID NO:1 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was used as TGFβ1, and human angiotensin II (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was used as AGT.

[0045] Telomere measurements of blastocyst-stage embryos were performed using real-time PCR. The cell number of the embryos to be used for the measurement was determined in advance by Hoechst staining, and embryos of the same developmental stage and cell number were selected for DNA extraction. Telomere length was calculated as the telomere length per blastomere.

[0046] Embryos cultured with TGFβ1 for 30 hours after fertilization showed improved quality (cell number) of blastocyst-stage embryos (Table 1). Furthermore, telomere length was significantly increased in TGFβ1-treated embryos (Figure 1).

[0047] [Table 1]

[0048] We also found that telomere length was significantly increased in AGT-treated embryos (Figure 2).

[0049] It was also confirmed that TGFβ1 has a similar telomere elongation effect in pig embryos.

Claims

1. A composition for in vitro elongation of chromosome telomeres in an embryo, comprising TGFβ1 or AGT.

2. The composition of claim 1 comprising TGFβ1.

3. The composition of claim 1 comprising AGT.

4. The composition according to claim 1 for culturing a fertilized egg or embryo within 48 hours after fertilization.

5. The composition of claim 1 , wherein the composition comprises follicular fluid.

6. The composition of claim 1, which increases the cell number of a blastocyst stage embryo during the development of the embryo.

7. The composition of claim 1 , which promotes development of the embryo.

8. The composition of claim 1 , wherein the embryo is a bovine embryo.

9. The method comprises culturing a fertilized egg or embryo in vitro in the presence of the composition according to any one of claims 1 to 8. A method for producing embryos with extended chromosomal telomeres.

10. An embryo produced by the method of claim 9.

11. A recipient livestock animal having the embryo according to claim 10.

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