WO2010021392A1 - In vitro fluorescence observation method for mammal embryo, and method for selecting mammal embryo reduced in risk of implantation failure or abortion - Google Patents

Abstract

Disclosed is an in vitro fluorescence observation method for a mammal embryo, which comprises obtaining fluorescence images of the mammal embryo that has been fluorescently labeled over a period of time by means of a confocal fluorescence microscope equipped with a laser light source and a Nipkow-disk type confocal unit.  Also disclosed is a method for selecting a mammal embryo that is reduced in the risk of implantation failure or abortion by utilizing the in vitro fluorescence observation method.

Description

Method for in vitro fluorescence observation of mammalian embryo and method for selecting mammalian embryo with low risk of implantation failure or miscarriage

The present invention relates to a fluorescence observation method for a mammalian embryo without damage to embryo development. The present invention also relates to a method for selecting a mammalian embryo with a low risk of implantation failure or miscarriage.

In Japan and other advanced countries, the declining birthrate is a serious problem. This is not only due to social factors, but also an increase in infertility. Currently, couples suffering from infertility are said to be 1-1.5 out of 10 pairs, which is not necessarily a minor symptom. Therefore, assisted reproduction (Artificial Reproductive Technology, ART) such as in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) has been developed and implemented, but implantation failure and miscarriage still occur frequently. is the current situation. These causes include embryonic chromosomal abnormalities in addition to the technical problems of ART. Furthermore, in recent years, “single blastocyst transfer” in which only one embryo is transferred has been strongly recommended in order to avoid the risk to the mother due to multiple pregnancy. From such a background, in order to reduce miscarriage and improve the pregnancy rate, it is desired to develop a technique for diagnosing embryos before transplantation / implantation and selecting embryos that are safely and reliably generated.

As one of the preimplantation genetic diagnosis methods currently being carried out all over the world, a part of the blastomeres constituting polar bodies or advanced embryos are removed by biopsy and fluorescence in situ hybridization (FISH) method is used. There is a method for identifying an embryo having an abnormality in a chromosome (Non-patent Document 1). This method is very effective because chromosomal aneuploidy, such as trisomy (Chromosome triploid) in Down's syndrome, can be identified before implantation. On the other hand, chromosome aneuploidy is known to stop embryogenesis before and after implantation, and is considered one of the causes of implantation failure and miscarriage. Therefore, it is expected that only the embryos judged to be normal by this diagnostic method are transplanted into pseudopregnant animals, and as a result, the pregnancy rate is improved. However, it has been reported that FISH does not lead to an improvement in pregnancy rate because the result of FISH varies from blastomere to each other or the damage to the embryo of biopsy manipulation is high (Non-patent Document 2).

A method has been reported that uses electrophysiological techniques to measure the respiratory activity of individual embryos in a non-invasive manner and to use the correlation between respiratory activity and subsequent embryo development to select embryos with good development. (Non-patent Document 3). However, even if this method is used, it is difficult to determine the presence or absence of a chromosomal abnormality.

A technique for observing the shape and position of a spindle in an ovum without staining using an optical technique has been reported, and attempts have been made to use this technique for selection of embryos (non-patent literature). 4).

In order to complete embryo selection technology, in order to eliminate the adverse effects on embryo development after observation, it is necessary to develop a method for observing early embryos in detail while suppressing damage to the embryo as much as possible. It is essential. In particular, a technique capable of detecting a chromosomal abnormality that is the main cause of miscarriage is effective.

The present inventors have been developing fluorescent imaging techniques that can observe various phenomena observed during early embryogenesis alive, and apply mRNA encoding fluorescent proteins to unfertilized eggs and embryos immediately after fertilization. We have reported a live cell imaging technique for embryos characterized by microinjection, incubating the embryo for several hours, then transferring it to a culture apparatus on a fluorescence microscope, and observing the temporal change in the signal obtained (non- Patent Document 5).

However, in this method, as a fluorescent signal detection means, a laser beam is scanned by a fluorescent microscope using a mercury lamp or a xenon lamp as a light source, or a scanning type (for example, a galvanoscan device using a galvanomirror) to the object to be observed. A confocal laser microscope is used. According to further studies by the inventors, these methods result in too much light hitting the sample and / or too long the exposure time, causing embryogenesis to stop during the imaging, or death of the embryo, Although in situ observation is possible, it has been found that individuals cannot be generated from the embryo after long-term fluorescence observation.

Although several groups have already reported on live cell imaging technology for early embryos before implantation, there are very few examples of successful detailed live cell imaging without adverse effects on embryo development after observation.

Non-Patent Document 6 reports that mitochondrial fluorescence was observed in hamster embryos using a two-photon excitation microscope, and that offspring were obtained from the embryos.

The object of the present invention is to develop a method for observing chromosome dynamics in early embryos in detail while suppressing damage to the embryo as much as possible. A further object of the present invention is to apply this method and develop a technique for selecting embryos that are safe and reliably generated with good development.

In order to solve the above-mentioned problems, various conditions in an embryo fluorescence imaging test were examined.
First, the concentration conditions of mRNA encoding a fluorescent protein used for detection of chromosomes to be injected into embryos were examined. As a result, it was found that if a specific concentration range of mRNA is injected into an embryo, a sufficient fluorescence signal can be obtained and the birth rate is not reduced.
Subsequently, the signal detection device was improved. A normal inverted microscope is used as the microscope, but in order to obtain detailed Z-axis information without blur, a confocal image using a laser device as an excitation light source is acquired. In order to reduce the amount of light striking the embryo to the limit, a nippo disc confocal unit is used to irradiate the target embryo with laser light through a hole in the rotating nippo disc for a very short time. An ultra-sensitive EM-CCD camera was attached to obtain the maximum possible. Equipped with an automatic control filter to enable multicolor observation, and by combining an electric XY stage and Z-axis motor, three-dimensional observation and simultaneous observation of multiple embryos, that is, a total of 6 dimensions (X-axis, Y-axis, Z-axis, time, multicolor, multisample) can be imaged.
In addition, various experimental conditions such as the amount of emitted laser light and observation time were set in detail to eliminate as much as possible the adverse effects on embryo / ontogenesis. As a result, 6-dimensional observation was performed for about 70 hours from the 1-cell stage to the blastocyst stage, and even after taking a total of about 50,000 fluorescent photographs during this period, individuals were successfully generated from the embryo without any problems. did.

Furthermore, by applying the above imaging technique to mouse ICSI embryos and observing the chromosome dynamics in the first somitosis that transitions from the 1-cell stage to the 2-cell stage, embryos that are at high risk of miscarriage in the future Was identified by the 2-cell stage, and the embryo was excluded from the transplantation, and as a result, the pregnancy rate was successfully improved. Based on the above findings, the present invention has been completed.

That is, the present invention relates to the following.
[1] A method for in vitro fluorescence observation of a mammalian embryo, comprising acquiring fluorescence images of a fluorescently labeled mammalian embryo over time with a confocal fluorescence microscope equipped with a laser light source and a nipou disc confocal unit.
[2] A fluorescently labeled mammalian embryo is obtained by injecting RNA into the embryo that encodes the fluorescent protein and can be translated into the fluorescent protein in the embryo. [1] The method described.
[3] The method according to [2], wherein the amount of RNA to be injected is a sufficient amount for obtaining a fluorescence image, and is 2.5 pg or less per embryo.
[4] The method according to [1], wherein fluorescence images of mammalian embryos are continuously acquired at a time interval of at least 3.75 minutes.
[5] The method according to [1], wherein a fluorescent image of the mammalian embryo is acquired over time for at least 20 hours.
[6] At the time of one observation, the focal point of the confocal fluorescence microscope is moved with respect to the embryo in the height direction with reference to the stage surface of the microscope at an interval of 3 μm or less, and the embryos in each layer The method according to [1], wherein a fluorescent image of the cross section is obtained.
[7] A method for selecting a mammalian embryo having a low risk of implantation failure or miscarriage, comprising the following steps:
(I) preparing a mammalian embryo that expresses a fusion protein of a chromosome constituent protein and a fluorescent protein or a fusion protein of a spindle constituent protein and a fluorescent protein;
(II) Obtaining a fluorescence image of the mammalian embryo obtained in (I) over time by a confocal fluorescence microscope equipped with a laser light source and a Niipou disc type confocal unit; and (III) obtained in (II) Based on the obtained fluorescence image, an embryo that does not exhibit abnormal chromosomal morphology is selected as a mammalian embryo having a low risk of implantation failure or miscarriage.
[8] The mammalian embryo of step (I) encodes a fusion protein of a chromosome constituent protein and a fluorescent protein or a fusion protein of a spindle constituent protein and a fluorescent protein, and is translated into the fusion protein in the embryo The method according to [7], which is prepared by injecting the obtained RNA into the embryo.
[9] The method according to [7], wherein the mammalian embryo of step (I) is a 1-cell stage or 2-cell stage embryo.
[10] The method according to [7], wherein the mammalian embryo is produced by intracytoplasmic sperm injection (ICSI) or circular sperm cell injection (ROSI).
[11] The method according to [7], wherein the abnormal chromosome morphology is abnormal chromosome segregation during somatic cell division.

By using the method of the present invention, it is possible to observe the early embryo in detail while suppressing damage to the embryo as much as possible. By using the method of the present invention, it is possible to continuously observe fluorescence while keeping a mammalian embryo alive, and furthermore, the embryo can be individually generated without any problem. And the subsequent ontogeny can be directly linked and evaluated. Furthermore, by observing chromosome dynamics during the first somatic cell division using the method of the present invention, it is possible to identify embryos at a very early stage, up to the 2-cell stage, at a high risk of miscarriage in the future. It becomes. And it becomes possible to improve a pregnancy rate significantly by removing the identified abnormal embryo from the transplant object.

It is a simple schematic diagram for demonstrating the structure of [the confocal fluorescence microscope provided with a laser light source and a Niipou disc-type confocal unit] used by this invention. Imaging safety assessment for pre-implantation diagnosis. a) Schematic diagram of safety evaluation. b) Typical time-lapse image of spindle (green tubulin) and nucleus (red H2B) dynamics during pre-implantation development. An image selected every 2 hours from a series of images obtained every 7.5 minutes is shown. The time after the start of observation is shown below the panel. c) Newborn born after long-term 6D imaging. d) A chart showing changes in the weight of the child. They were created from embryos exposed to three intensities (0, 20, 70 mW) of laser light at intervals of 7.5 minutes for about 70 hours (details are given in Table 2). The horizontal and vertical axes indicate the week after birth and the weight, respectively. “N” means the number of mice analyzed. e) Child fluorescence image. An asterisk indicates a transgenic mouse expressing high sensitivity green fluorescent protein (EGFP) as a positive control. f) Typical electrophoresis pattern for genotyping analysis of tail tip DNA. Lanes 1-22 are from embryos of the imaged embryo; Lane N is from a normal BDF1 strain mouse as a negative control; P is from an EGFP-expressing mouse as a positive control; and M is a DNA size marker. g) Imaging device. A normal inverted microscope equipped with a Nipkow disc confocal unit, EM-CCD camera, Z-axis motor and automatic XY-axis stage. All of these were controlled using MetaMorph imaging software. Embryos were cultured in a CO ₂ incubator on stage. Details are shown in FIG. FIG. 2 is an original color photograph. Fluorescence microscopy of embryos injected with various concentrations of mRNA. Various combinations of equal amounts of mRNA encoding EGFP-α-tubulin (tubulin, green) and mRNA encoding H2B-mRFP1 (H2B, red) were added to the second meiotic / end-stage IVF embryo. At a concentration of 0-250 ng / μL. After 70 hours of incubation, they were observed using an imaging system. NI means “non-injection”. FIG. 3 is an original color photograph. A device used to evaluate the safety of imaging. FIG. 4 is an original color photograph. Abnormal chromosome segregation in embryos created by intracytoplasmic sperm injection (ICSI). (Ac) Time-lapse images of normal chromosome segregation (NCS; a) and abnormal chromosome segregation (ACS; b, c) at the first somatic division of an ICSI embryo. Keywords: m, male pronucleus: f, female pronucleus; pb, polar body. (Df) Immunostaining of 2-cell embryos with ACS. Histone H2B-mRFP1 mRNA (red) was injected into the embryo, and anti-lamin B antibody (lamin B; d), anti-phosphorylated histone H3 Ser10 antibody (pH3 ser10; e) and anti-phosphorylated H2A. Stained with X antibody (pH 2A.X; f). Arrows indicate chromosome fragments that are abnormally separated during the process of somatic cell division. Arrowheads indicate micronuclei. FIG. 5 is an original color photograph. Abnormal chromosome segregation occurs before and after implantation. (A, b) Long-term imaging of embryos with ACS. (A) Embryo with ACS that is developing to morula. (B) Embryo stopped after the 4-cell stage. (C, d) Different staining patterns of blastocysts obtained from NCS embryos (c) and ACS embryos (d) treated with anti-Oct3 / 4 antibody (green) and anti-Cdx2 (red). (E) Schematic diagram of evaluation of ICSI embryos screened by live cell imaging. (F) E7.5 uterus of pseudopregnant female mice transplanted with NCS (left) and ACS (right) embryos. The arrow indicates the landing site. (G) Decidual membrane of NCS and ACS embryos at E7.5 and E9.5. The arrow indicates the embryo removed from the decidua. FIG. 6 is an original color photograph. Optical microscope observation image of ACS and NCS2 cell embryos after separation for live cell imaging. FIG. 7 is an original color photograph. Frequency of abnormal chromosome segregation in embryos created by circular sperm cell injection into normal oocytes and intracytoplasmic sperm injection with Kid ^{− / −} mouse oocytes. Normal (upper) and abnormal chromosome segregation (lower) in round sperm cell injection (ROSI) embryos (a) and Kid ^{− / −} mutant ICSI-produced embryos (b). Kid ^{− / −} female oocytes were microinjected with fresh sperm from a sexually mature BDF1 male. Keywords: m, male pronucleus; f, female pronucleus; pd, polar body. Arrows indicate abnormally separated chromosomes and micronuclei. FIG. 8 is an original color photograph. Nucleus / chromosome abnormality observed in monkey microinsemination embryonic somatic cell division. (A) Chromosome partitioning abnormality frequently observed in mouse microinseminated embryos (see FIGS. 5b and c). Part of the chromosome was misplaced during distribution (arrow). (B) Multinucleated embryo. An abnormality in which two or more nuclei are formed in the two-cell stage without having a single chromosome after distribution. (C) Embryo showing chromosome fragmentation. Chromosomes are not already aligned in the equator plane of the spindle in the metaphase (metaphase), and the chromosomes are dispersed after distribution to both poles (arrows). As a result, countless nuclear and chromosomal signals are observed in the 2-cell stage. Is done.

(1. In vitro fluorescence observation method of mammalian embryo)
The present invention relates to a method for in vitro fluorescence observation of a mammalian embryo (this book), comprising acquiring fluorescence images of a fluorescently labeled mammalian embryo over time by a confocal fluorescence microscope equipped with a laser light source and a nipou disc confocal unit. Inventive method I) is provided.

The embryo used in the method I of the present invention is derived from a mammal. Examples of mammals include, for example, laboratory animals such as rodents and rabbits such as mice, rats, hamsters, and guinea pigs, domestic animals such as pigs, cows, goats, horses, sheep and minks, pets such as dogs and cats, humans, Examples include, but are not limited to, primates such as monkeys, cynomolgus monkeys, rhesus monkeys, marmosets, orangutans and chimpanzees.

Mammalian embryos can be generated in vitro using reproductive techniques known per se, such as in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), circular sperm cell injection (ROSI), and the like. is there.

In the method I of the present invention, any developmental stage mammalian embryo can be used as an observation target. By using the method I of the present invention, it is possible to carry out detailed fluorescence observation of the embryo while minimizing damage to embryo development, and the embryo after observation can be used as a pseudopregnant mammal uterus or oviduct When transferred to, normal implantation will occur and offspring derived from the embryo will be born. Therefore, the method I of the present invention is advantageous for observing embryos in a preimplantation stage, particularly embryos that are scheduled to be transferred to the uterus and oviduct of a pseudopregnant mammal in the future. Examples of embryos at the stage before implantation include embryos at the 1-cell stage, 2-cell stage, 4-cell stage, 8-cell stage, morula stage, blastocyst stage, etc., but are not limited thereto. It is not a thing.

The mammalian embryo used in the method I of the present invention is fluorescently labeled by a desired method so that it can be detected by a fluorescent microscope. Examples of the fluorescent labeling method include expression of fluorescent protein in the embryo, staining with a fluorescent substance (for example, FITC) binding antibody that specifically recognizes the antigen in the embryo, injection of a fluorescent nucleic acid probe, and the like. However, it is not limited to these. As fluorescent proteins, those known per se, for example, GFP, RFP, CFP, YFP, and their improved variants (eg, EGFP, EYFP, etc.) can be used. The fluorescent protein of the color can be selected. In addition, by expressing a fusion protein of a desired protein and the fluorescent protein in the embryo, the technology for observing the localization of the protein in the embryo and the morphology of the in-embryonic microtissue where the protein is localized is observed. Widely known, such fluorescent fusion proteins are also useful for fluorescent labeling of mammalian embryos.

In one preferred embodiment, the embryo is fluorescently labeled by expressing in a mammalian embryo a fusion protein of a chromosome constituent protein and a fluorescent protein or a fusion protein of a spindle constituent protein and a fluorescent protein. When a fusion protein of a chromosomal component protein and a fluorescent protein is expressed in a mammalian embryo, the fluorescent fusion protein is localized in the chromosome, so that a detailed fluorescent image of the morphology and structure of the chromosome of the mammalian embryo can be obtained. Is possible. In addition, when a fusion protein of a spindle protein and a fluorescent protein is expressed in a mammalian embryo, the fluorescent fusion protein is localized throughout the spindle and the cytoplasm. Can be traced in detail by fluorescence observation. Further, by using a combination of a fusion protein (A) of a chromosomal component protein and a fluorescent protein and a fusion protein (B) of a spindle component protein and a fluorescent protein, the dynamics of the chromosome in the embryo development process can be determined. It becomes possible to track in detail over time through time and at rest. When using the combination, the emission wavelength of the fluorescent protein contained in the fusion protein (A) is different from the emission wavelength of the fluorescent protein contained in the fusion protein (B) so that the two types of fusion proteins can be distinguished. Is preferred. In the detection of the chromosome segregation abnormality described below, an embodiment in which the fusion proteins (A) and (B) are combined in this way is preferable, but the chromosome segregation abnormality can also be detected using only the fusion protein (A). Is possible.
As a chromosome constituent protein, histone (H1, H2A, H2B, H3, H4, etc.) can be used, but is not limited thereto.
As the spindle component protein, tubulin (α, β) or the like can be used, but is not limited thereto.

In addition to the above, a fusion protein of a protein having a property of binding to the chromosome itself or its constituent components and a fluorescent protein (for example, EGFP-MBD-, which is a fluorescent probe that binds to methylated DNA previously developed by the present inventor) NLS (Gensis, 43, 71-79 (2005))) can be used, but is not limited thereto.

Methods for expressing fluorescent proteins in mammalian embryos include the following:
(A) introduction of an expression vector capable of expressing a fluorescent protein in a mammalian embryo into the mammalian embryo;
(B) injection of RNA encoding a fluorescent protein and capable of being translated into the fluorescent protein in a mammalian embryo into a mammalian unfertilized egg or mammalian embryo; and (C) the fluorescent protein gene is in the embryo Use of embryos from transgenic mammals integrated into the genome in an expressible manner.

The method (A) is one of the most popular methods. However, since expression vectors (plasmid vectors, viral vectors, etc.) usually contain DNA, the introduced DNA is integrated into the genome of the mammalian embryo. As a result, a transgenic mammal may be generated. On the other hand, in the method (B), RNA is directly injected into an embryo, so that there is theoretically no risk of producing a transgenic mammal. Therefore, when the generation of a transgenic animal from a mammalian embryo to be observed is not desired (for example, use in the medical or livestock field), the method (B) is advantageous.

The RNA (mRNA) used in the method (B) includes a translation initiation region on the 5 'side of the coding region of the fluorescent protein so that it can be translated into the fluorescent protein in the mammalian embryo. The RNA may also contain a cap structure at the 5 'end so that it can be stabilized in the embryo and translated efficiently. It is also preferred that the RNA has a poly A region at the 3 'end so that the RNA can be stabilized in the embryo and translated efficiently. The length of the poly A is usually 65 or more, preferably 80 or more (for example, 83). The RNA used in the method (B) is the DNA of the target fluorescent probe in the plasmid (pcDNA3.1-poly (A83), Gensis, 43, 71-79 (2005)) previously developed by the inventors. Can be obtained by in vitro transcription using a commercially available kit using this as a template, and isolating and purifying the RNA fraction (mRNA) from the transcript. To avoid the risk of transgenic animal development due to DNA contamination, the transcript is preferably subjected to DNase treatment. The RNA used in the method (B) may be produced by chemical synthesis. For the specific preparation method of RNA used in the method (B), see Gensis, 43, 71-79 (2005).

In the method (B), RNA may be injected into either an unfertilized egg or embryo of a mammal. When embryos are created in vitro (eg IVF, ICSI, ROSI, etc.), embryos often die during the process of creating embryos. Therefore, fertilization is performed rather than injecting RNA into unfertilized eggs. It is preferable to inject RNA into the surviving embryo later because the labor of the injection operation can be reduced. When RNA is injected into an unfertilized egg, an insemination operation with a male gamete (sperm, sperm cell, etc.) must be performed thereafter.

In the method (B), an amount of RNA sufficient to obtain a fluorescence image of a mammalian embryo by a fluorescence microscope and with minimal damage to embryo development is injected into an unfertilized egg or embryo. The Specifically, as the amount of RNA sufficient for obtaining a fluorescence image,
For example, 0.1 pg (equivalent to 10 ng / μl × 10 picoliter) or more per embryo is exemplified.
Specifically, the amount of RNA in which damage to embryonic development is suppressed is as follows:
2.5 pg per embryo (250 ng / μL × 10 picoliter equivalent) or less,
Preferably, 0.5 pg (50 ng / μL × 10 picoliter equivalent) or less is exemplified.
Therefore, in the method (B),
Usually 0.1pg to 2.5pg,
Preferably 0.1pg to 0.5pg
Of RNA is injected into one unfertilized egg or embryo.

After the RNA injection, the mammalian embryo is cultured for 3 to 5 hours in an appropriate medium in order to translate a sufficient amount of fluorescent protein to obtain a fluorescent image, and then subjected to observation with a confocal microscope.

Hereinafter, observation of fluorescently labeled mammalian embryos using a confocal microscope will be described in detail.

In Method I of the present invention, a fluorescently labeled mammalian embryo is observed with a confocal fluorescence microscope equipped with a laser light source and a Niipou disc confocal unit, and a fluorescence image is acquired over time.

In order to allow long-term fluorescence observation, the mammalian embryo is placed in a drop of a suitable medium (eg, CZB medium) on a suitable culture vessel (eg, glass bottom dish). As the culture vessel, a culture vessel made of a member that transmits excitation light and fluorescence is selected so as not to hinder fluorescence observation. In order to prevent evaporation of the medium, mineral oil is preferably overlaid on the medium drop. The culture vessel is placed in a microscope CO ₂ incubator placed on an observation table of a fluorescence microscope, and is at a temperature suitable for embryo development (eg 37 ° C., 38 ° C. for monkey embryos) and humidity (eg 100%). The embryos are cultured under conditions of CO ₂ concentration (eg, 5%).

One of the important features of the present invention is that a confocal fluorescence microscope using a Niipou disc confocal unit is selected from various fluorescence microscopes, and this is used for in vitro fluorescence observation of mammalian embryos and mammalian embryos. It is in the point applied to the selection of. This has enabled long-term three-dimensional analysis for the first time without adversely affecting embryogenesis.
FIG. 1 is a simple schematic diagram for explaining the configuration of a [confocal fluorescence microscope including a laser light source and a Niipou disc confocal unit] used in the present invention. In the figure, in order to explain the principle, detailed depiction of the optical system is omitted.
As shown in FIG. 1, the laser light source 1 is an excitation light source for exciting a fluorescent substance. The Niipou disc type confocal unit 2 focuses the laser light L1 from the light source 1 on the observation object S through the Nipo disc that rotates at high speed, and focuses on the emitted light source of the emitted fluorescence L2. , An apparatus for sending the fluorescence L2 to the imaging device 3 through the nippo disk. More specifically, the confocal microscope (a) in the excitation light irradiation, a rotating nipou disk (also referred to as a Nipo plate, a Nipkow disc, or a pinhole plate) 22 which rotates laser light (excitation light) L1 from the light source 1 22 By passing through the through-hole provided in the laser beam, the observation object S is irradiated with the laser beam L1 while being focused at a predetermined depth locally. In (b) fluorescence imaging, the irradiation point The image pickup device 3 receives and synthesizes fluorescence as one pixel (or a scanning line in which the pixels are connected by movement of the through-hole) emitted from there. It is a microscope.

The Nipkow disk is a known scanning disk, and as shown in FIG. 1, a disk 22 configured to block the laser light is configured to be rotatable by a motor for rotation driving, and on the surface of the disk. Are provided with a large number of through holes so as to draw a spiral sequentially from the rotation center side to the outer peripheral side. Each through hole from the rotation center side to the outer peripheral side corresponds to each scanning line constituting the image. When the disk is rotated while irradiating a specific irradiation area of the Nipkow disk surface with the laser beam L1, the laser beam passes through the through-hole only when each through-hole crosses the irradiation area and is irradiated onto the observation object. The
A local image of an observation object obtained by irradiating a laser beam having a specific beam cross section through a focus through a through-hole (in the present invention, an image based on fluorescence emitted from the object, not just reflected light) Can be synthesized through focusing on the same through-hole, and a clear image with good contrast can be synthesized.

In the apparatus configuration of FIG. 1, the condensing disk 21 is provided on the laser light source side of the nipo disk so that the laser beam can preferably pass through the through hole (not shown in the figure) of the nipo disk 22. The condensing disk 21 is a disk that rotates integrally with the Nipkow disk 22, and a lens is provided on the plate surface at a position corresponding to the through hole of the Nipkow disk. Further, in the apparatus configuration of FIG. 1, the fluorescence L2 emitted from the observation object S returns again through the through-hole of the Nipkow disk 22 through which the excitation light has passed, and the light branching means (for example, a dichroic mirror inclined at 45 degrees) ) The direction is changed by 23 and the image pickup apparatus 3 is directed.
In excitation light irradiation and fluorescence imaging, not only one plane irradiation / imaging with a fixed depth of the irradiation point (focal point) (also referred to as a height with respect to the stage surface of the microscope), but also the depth. A three-dimensional image of the observation object can be obtained by sequentially irradiating and imaging a multilayer plane and combining the obtained plane pixels of each layer.
Such a Niipou disc confocal unit and a confocal fluorescence microscope technology for imaging fluorescence by irradiating a laser beam using the same are known per se. For example, for a confocal microscope using a Nipo disc, This is described in detail in JP-A-5-60980.
In order to preferably pass and change the optical path by the light branching means, the prior art may also be referred to as appropriate for the detailed configuration of the optical system, such as using polarized light for the laser light.

Specifications of Nipkow disk (diameter of disk, number of through holes (= number of scanning lines), distance of each through hole from the center of disk, diameter of through hole, rotation speed of disk) The specifications of the irradiated laser light (output, wavelength, beam diameter (that is, the irradiation area on the plate surface), and the shape of the irradiation area) can suppress damage to the embryo and enable preferable fluorescence observation. Thus, a preferable combination may be appropriately selected. A preferred example of the specifications of the Nipkow disk is the Nipkow disk scanning confocal unit (CSU10) manufactured by Yokogawa Electric Corp.
The number, the opening shape, and the opening area of the through holes of the Niipou disc may be determined in consideration of the damage to the embryo and the resolution of the target image. The through holes are provided in an arrangement pattern that draws a spiral on the plate surface at a position corresponding to the number of scanning lines corresponding to the number of scanning lines. Regarding the arrangement pattern of the through holes of the Niipou disc and the rotational speed of the disc, a known technique may be referred to within a range in which damage to the embryo can be suppressed.

The wavelength and output of the laser light may be selected so as to suppress damage to the embryo so as to be excitation light appropriate for the fluorescent substance and in association with the specifications of the Niipou disk as described above.
From the viewpoint that the wavelength of the laser light is excitation light of an available fluorescent substance, for example, those having a peak near 488 nm for EGFP and those near 561 nm for mRFP1 are preferable. Examples of a laser light source (laser device) that can emit laser light having such a wavelength include a krypton argon gas laser light source (emits light with wavelengths of 488 nm, 568 nm, 647 nm, and 752 nm). Various laser apparatuses that can emit light having a wavelength may be used.
Since krypton argon gas laser can oscillate laser light in a relatively wide wavelength range, it is possible to obtain many types of fluorescent signals with one laser device by labeling embryos with fluorescent probes of various excitation wavelengths. There are also benefits.
When a krypton argon gas laser is used as the laser light source and the specifications of the nippo disk and the irradiation area on the plate surface of the nippo disk are in the above range, the laser output is preferably 20 to 70 mW, particularly 20 to 25 mW. is there.
With these combinations, while irradiating laser light so that fluorescence observation is possible, damage to the embryo is suppressed, and final differentiation from embryo to offspring after long-term fluorescence observation becomes possible.
On the other hand, when the embryo is observed using a fluorescent microscope using a mercury lamp or xenon lamp as a light source, or a scanning confocal laser microscope that scans and sequentially irradiates laser light using a galvanoscan device, Although the exposed light is too intense or the exposure time is too long and can be observed, the embryo is damaged and cannot be terminally differentiated to offspring.

In order to minimize damage to embryonic development, fluorescent images are taken by “top shot” at intervals of a certain time or more. Usually, fluorescent images are continuously acquired at intervals of 3.75 minutes or more, preferably 7.5 minutes or more (for example, intervals of 3.75 to 60 minutes, preferably 7.5 to 30 minutes).
The duration of exposure of the embryo to the excitation light in one image is usually in the range of 100 ms to 500 ms, preferably 100 ms to 200 ms, depending on the laser output and the type of laser. Is within.

Also, in order to avoid oversight of minute chromosome fragments (for example, fragments of about 1 μm), the focal point is moved in the Z-axis direction (that is, the height direction with respect to the stage surface of the microscope) at any step interval. It is important how many sections of the embryo are acquired.
In other words, abnormal chromosome fragments observed during embryonic division are very small, about 1 to 3 μm. Therefore, when performing confocal observation, a minute interval in the Z-axis direction (for example, 2 as described later). If it is not photographed at intervals of 3 μm, there is a possibility of being overlooked. In addition, mammalian embryos are very thick cells (mouse embryos are about 100 μm, monkeys and human embryos are about 150 μm), so it is necessary to photograph nearly 50 images in the Z-axis direction to capture the whole. It becomes.
In view of the above, in the present invention, at one observation time point, in the present invention, an interval of, for example, 20 μm or less, preferably an interval of 10 μm or less, and more preferably 5 μm or less, in the Z-axis direction. And most preferably at intervals of 3 μm or less (for example, at intervals of 2 to 3 μm), changing the focal point to obtain confocal images of each layer, and obtaining a stereoscopic image with higher resolution. Is recommended.
For example, in an embryo with a diameter of 100 μm, if the focal point is moved at an interval of 2 μm in the Z-axis direction, a total of 51 confocal images (tomographic images) including the images of the uppermost layer and the lowermost layer will be acquired. Can do. By constructing a high-resolution three-dimensional image in the height direction, it is possible to observe minute chromosome fragments.

Since the intensity of the laser beam irradiated to the mammalian embryo to be observed is suppressed as much as possible, a detection device capable of detecting even weak fluorescence is used when acquiring a fluorescent image. Examples of such a detection device include an ultra-sensitive EM-CCD camera.

By installing an electric XY stage and a Z-axis motor in the microscope, it is possible to observe mammalian embryos in three dimensions and simultaneously observe multiple embryos.

Furthermore, by installing excitation and absorption filter wheels respectively on the laser output side and immediately before the camera, multicolor observation can be performed by automatic control.

Furthermore, automatic device control and image processing become possible by connecting the above-mentioned devices to a computer.

By using the method I of the present invention, while minimizing damage to embryonic development, for a long period (for example, 20 hours or longer, preferably 40 hours or longer, more preferably 50 hours or longer, even more preferably 60 hours or longer). , More preferably 70 hours or more), it is possible to continuously observe the fluorescence of the mammalian embryo. For example, embryo development from the 1-cell stage to the 2-cell stage (about 20 hours in mice), the 4-cell stage (about 40 hours in mice, about 60 hours in monkeys) and the blastocyst stage (about 70 hours in mice) After continuous fluorescence observation in vitro, when the embryo is transferred to the uterus or oviduct of a pseudopregnant mammal female, the embryo is normally implanted and a child derived from the embryo is born.

By using the method I of the present invention, it is possible to continuously observe the fluorescence while keeping the mammalian embryo alive, and further, the embryo can be generated individually without any problem. It is possible to directly link and evaluate the relationship between the phenomenon and subsequent ontogeny.

(2. Method of selecting mammalian embryos with low risk of implantation failure or miscarriage)
Based on the advantages of Method I of the present invention as described above, the present invention further provides a method for selecting a mammalian embryo with a low risk of implantation failure or miscarriage (Method II of the present invention) comprising the following steps:
(I) preparing a mammalian embryo that expresses a fusion protein of a chromosome constituent protein or a spindle constituent protein and a fluorescent protein;
(II) Obtaining fluorescence images of a fluorescently labeled mammalian embryo over time by a confocal fluorescence microscope equipped with a laser light source and a Niipou disc type confocal unit; and (III) Fluorescence images obtained in (II) Based on the above, an embryo that does not exhibit an abnormal chromosomal morphology is selected as a mammalian embryo with a low risk of miscarriage.

The definition of terms in the method II of the present invention is the same as that of the method I of the present invention.

The operation of step (I) is performed according to the method described in the section (1. In vitro fluorescence observation method of mammalian embryo).

In order to suppress the development risk of transgenic animals, in step (I), a fusion protein of a chromosomal component protein and a fluorescent protein or a fusion protein of a spindle component protein and a fluorescent protein is encoded, and By injecting RNA that can be translated into a fusion protein into the embryo, a mammalian embryo that expresses a fusion protein of a chromosome constituent protein and a fluorescent protein or a fusion protein of a spindle constituent protein and a fluorescent protein is prepared.

In step (I), in order to achieve detailed analysis of chromosome morphology, it is preferable to use a combination of a fusion protein of a chromosome constituent protein and a fluorescent protein and a fusion protein of a spindle constituent protein and a fluorescent protein. . As a result, the dynamics of the chromosomes during the embryonic development process can be traced in detail over time during cleavage and at rest.

Prepared in step (I) because the size of the cells that make up the embryo is large, RNA injection is easy, and there is no overlap between cells in the field of view, allowing detailed observation of chromosome morphology. The mammalian cell embryo to be used is preferably an unfertilized ovum or a 1-cell stage or 2-cell stage embryo, most preferably a 1-cell stage embryo.

The operation of the step (II) is performed according to the method described in the section (1. In vitro fluorescence observation method of mammalian embryo).

In step (III), based on the fluorescence image obtained in step (II), an embryo that does not exhibit an abnormal chromosomal morphology is selected as a mammalian embryo with a low risk of miscarriage.

Examples of abnormal chromosome forms include the following:
(A) Abnormal chromosome segregation during somatic cell division (B) Chromosome aneuploidy such as trisomy.

Embryos with such abnormal chromosomal morphology cannot be developed normally, even if they develop in vitro to the stage of morula and blastocyst, and even after implantation into pseudopregnant females, Almost everything is miscarried. Therefore, by eliminating embryos that have observed abnormal chromosome morphology by fluorescence observation, the remaining embryos (that is, embryos that do not exhibit abnormal chromosome morphology) are obtained as embryos with a low risk of implantation failure or miscarriage. be able to.

Specific examples of (A) include the following:
i) Separation of chromosomes accompanying somatic cell division, such as “forgetting” some chromosomes between chromosomes. Misplaced chromosomes remain floating in the cytoplasm after somatic cell division.
ii) A phenomenon in which some chromosomes protrude when male and female chromosomes are grouped together in a metaphase plate. Chromosomes that protrude in this way can be incorporated into one nucleus during somatic cell division.
iii) A combination of i) and ii).

In addition, the frequency of abnormal chromosome segregation during somatic cell division is significantly higher in embryos produced by ICSI or ROSI than in embryos obtained by IVF, which is a lower rate of successful birth after ICSI or ROSI. For this reason, the method II of the present invention is advantageous particularly when selecting an embryo with a low risk of implantation failure or miscarriage from embryos produced by ICSI or ROSI.

The mammalian embryo obtained by the method II of the present invention has been observed for fluorescence under the condition that damage to the embryogenesis is suppressed to a minimum, and further, an abnormal chromosomal morphology and an embryo with a high risk of miscarriage are removed. Therefore, by transferring this embryo to the uterus or oviduct of a pseudopregnant female, offspring derived from the embryo are born with high efficiency. Therefore, the method II of the present invention is useful as a technique for preventing implantation failure and miscarriage due to assisted reproduction (Artifical Reproductive Technology, 補助 ART) and improving the pregnancy rate in the fields of reproductive medicine and livestock.

Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to the examples shown below.

Example 1
[Method]
Collection of gametes Female BDF1 mice (7) by intraperitoneal injection of 5 IU pregnant mare serum gonadotropin (PMSG) and 5 IU human chorionic gonadotropin (hCG) (Imperial Organ, Tokyo, Japan) at 48 hour intervals. -12 weeks old) were superovulated. Oocytes with cumulus cells were harvested 13-15 hours after hCG injection. Sperm were harvested from the epididymis tail of BDF1 male mice (> 12 weeks) into 0.2 mL drops of TYH medium to obtain fertility by incubation at 37 ° C. for 2 hours under 5% CO ₂ . Freeze-thawed sperm was prepared by freezing sperm that acquired fertilizing ability in TYH medium (Jpn J Anim Reprod, 16, 147-151, 1971) at −25 ° C. and thawing immediately before use.

In vitro fertilization (IVF)
Oocytes with cumulus cells were collected in 0.2 mL of TYH medium and fertilized with sperm that acquired fertility (final concentration 100 / μL). After incubation for 2 hours at 37 ° C. under 5% CO ₂ , cumulus cells were dispersed by a short treatment with hyaluronidase (type IS, 150 units / mL, Sigma-Aldrich, St Louis, MO, USA).

Intracytoplasmic sperm injection (ICSI)
Second meiotic metaphase II (MII) oocytes were prepared by brief incubation in Chatot-Ziomek-Bavister (CZB) medium (Biol Reprod, 42, 432-440, 1990) containing hyaluronidase. 1 microliter suspension of sperm that has acquired fertilizing ability in TYH medium contains 12% (w / v) polyvinylpyrrolidone (PVP, Wako Pure Chemical Industries, Ltd, Osaka, Japan) in a micromanipulation chamber Mixed with 9 μL of HEPES buffered CZB medium. The head of each sperm was separated from the tail by applying a pulse to the head-tail junction by means of a piezo-driven pipette (PrimeTech, Ibaraki, Japan). Only the sperm head was injected into the cytoplasm of each MII oocyte.

Round sperm cell injection (ROSI)
To collect cells in the process of spermatogenesis, testicular seminiferous tubules from BDF1 male mice were chopped with scissors and suspended in HEPES buffered CZB medium. When the medium became cloudy, the cell suspension was filtered with Kimwipe to remove cell debris. In a micromanipulation chamber, 1 μL aliquots of spermatogenic cell suspension were mixed with 10 μL of 12% PVP / HEPES-CZB. MII oocytes were artificially activated by incubation for 20 minutes in Ca ²⁺ -free CZB medium containing 5 mM SrCl ₂ . Approximately 60 minutes after activation, circular sperm nuclei were injected into each oocyte using a piezo-driven micromanipulator.

Synthesis of mRNA in vitro After linearization of the template plasmid at the XbaI (EGFP-α-tubulin and H2B-mRFP1) or XhoI (EGFP-β-actin) sites, RiboMAX ™ Large Scale RNA Production Systems-T7 (Promega, MRNA was synthesized using Madison, WI, USA. For efficient translation of the fusion protein in the embryo, the 5 'end of each mRNA was capped using Ribo m7G CapAnalog (Promega) according to the manufacturer's protocol. In order to avoid incorporation of template DNA into the embryo genome, the reaction mixture for in vitro transcription was treated with RQ-1 RNase-free DNase I (Promega). The synthesized RNA was treated with phenol-chloroform and subsequently subjected to ethanol precipitation. After dissolution in RNase-free water, the mRNA was subjected to gel filtration using a MicroSpin ™ G-25 column (Amersham Biosciences, Piscataway, NJ, USA) to remove unreacted substrate and stored at −80 ° C. until use. .

Each mRNA synthesized by mRNA microinjection was diluted to an appropriate concentration using Milli-Q ultrapure water (Millipore Corp., Madison, Wis., USA), and the aliquot was placed on a micromanipulation chamber. Transfer the second meiotic / end-stage oocyte (approximately 2 hours after insemination or activation) to the HEPES-CZB medium on the chamber and use a piezo manipulator with a thin glass pipette (1-3 μm in diameter). mRNA was injected. The mRNA solution was once sucked into a pipette, and the zona pellucida and cell membrane were broken by applying a piezo pulse to the oocyte. 5-10 picoliters of solution was introduced into the oocyte and the pipette was quickly removed. Embryos injected with mRNA were incubated at 37 ° C. in a 5% CO ₂ atmosphere, and mRNA was translated until sufficient imaging was possible. Over 200 oocytes could be injected per hour and the survival rate after mRNA injection was close to 100%.

Transfer embryos to 5 μL drops of CZB medium on live cell imaging glass bottom dish (12 embryos / drop), incubator on microscope stage to 2 cell stage (about 20 hours) or blastocyst stage (about 70 hours) (Tokai Hit, Shizuoka, Japan). Unless otherwise specified, excitation light of two colors (green and red) was applied with a laser output of 25 mW, and 51 images (in 2 μm increments) were traced at 15-minute intervals in the Z-axis direction. A Nipow disk scanning confocal unit (CSU10, Yokogawa Electric Corp.) and an EM-CCD camera (iXON BV-887, Andor) were attached to an inverted microscope. By installing excitation and absorption filter wheels (Ludl) just before the laser output side and the camera, multi-color observation is possible by automatic control. By attaching a Z-axis motor (Mac5000, Ludl Electronic Products), 3D observation is possible. The imaging device has an automatic XY stage that can monitor a large number of embryos (usually imaged over 100 embryos) in a single assay. Control of the apparatus and image analysis were performed using MetaMorph software (Universal Imaging). A KrAr (krypton argon) gas laser (Melles Griot) was used as a laser light source. Regarding the laser output, the total output of the laser light source body was adjusted while measuring the respective light amounts of 488 nm and 561 nm emitted from the lens using Yokogawa's TB200 Optical power meter. The detector set is installed in a dark room where the temperature is kept around 30 degrees, and the temperature of the CO ₂ incubator for microscope (Tokai Hit, MI-IBC) is set so that the temperature of the observed medium is 37 ° C. It was. 5% CO ₂ gas was introduced into the incubator at a flow rate of 160 ml / min.

Embryo transplanted embryos transplanted with 2-cell stage embryos into the oviduct of a surrogate mother (ICR line) on day 0.5 of pseudopregnancy after mating with ligated ICR mice. Mulberry embryos and blastocysts were transplanted into the mother's oviduct on day 0.5 of pseudopregnancy or the mother's womb on day 2.5 of pseudopregnancy.

Plasmid Construction EGFP-α-tubulin (Yamagata K. et al., Genesis, 43, 71-79, 205) and histone H2B-mRFP1 (Yamazaki T. et al.) Inserted into pcDNA3.1 poly (A83) vector. , Reprod Dev, 53, 1035-1041, 2007), an mRNA plasmid was constructed as described previously. EGFP-β-actin was made by fusion of EGFP to the N-terminus of the full-length mouse β-actin cDNA. In order to isolate β-actin cDNA, PCR amplification was performed using a single-stranded cDNA derived from mouse testis as a template and the following primer set.
5'-TGGATCCCGAGCTATGGATGACGATATCGCTGCGCTG-3 '(SEQ ID NO: 1)
5'-AAGCGGCCGCCTAGAAGCACTTGCGGTGCACGAT-3 '(SEQ ID NO: 2)
The amplified fragment was co-inserted with the EGFP fragment into the EcoRI and NotI sites of the pcDNA3.1-poly (A83) vector.

Cutting the tail tip genotyping Son, once frozen. These were placed in lysis buffer (50 mM Tris / HCl pH 7.6, 0.1 M NaCl, 20 mM EDTA, 1% SDS, 0.1 mg / mL proteinase K and 0.08 mg / mL RNase A) overnight at 50 ° C. Incubated. The extracted chromosomal DNA was treated twice with a phenol-chloroform mixture and precipitated with ethanol. After dissolving in TE, the DNA concentration was adjusted to 0.3 μg / μL. One microliter of DNA solution was used for 12.5 μL of each reaction mixture for PCR analysis (Ex Taq, TaKaRa, Kyoto, Japan). PCR was performed under conditions of 35 cycles of 95 ° C. for 10 seconds, 58 ° C. for 30 seconds, and 72 ° C. for 60 seconds. The primer sets used are as follows.
(For amplification of translation region of EGFP)
5'-AATCTAGAATGGTGAGCAAGGGCGAG-3 '(SEQ ID NO: 3)
5'-AATCTAGACTTGTACAGCTCGTCCATG-3 '(SEQ ID NO: 4)
(For amplification of β-actin gene as a PCR control)
5'-AAAAGCTTGGCGCTTTTGACTCAGGA-3 '(SEQ ID NO: 5)
5'-GGAATTCAAGTCAGTGTACAGGCCAG-3 '(SEQ ID NO: 6)
Chromosomal DNA from B6D2F1 and GOF18-GFP transgenic mice (Dev Growth Differ, 41, 675-684, 1999) were used as negative and positive controls, respectively.

Embryos created by ICSI using immunostained freeze-thawed sperm were fixed with 4% paraformaldehyde (in PBS) at the 2-cell stage or blastocyst stage, and 0.25% Triton X100 / PBS Permeabilized. For blastocysts, the zona pellucida was peeled using an acidic tyrode solution before fixation. After blocking with 3% BSA / PBS containing 0.01% Tween-20, anti-Oct3 / 4 antibody (sc-9081, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Cdx2 antibody (CDX2-88, BioGenex , San Ramon, CA, USA), anti-lamin B antibody (sc-6217, Santa Cruz Biotechnology), anti-phosphorylated histone H3 antibody (Ser 10) (# 9701, Cell Signaling Technology, Danvers, MA, USA), or anti Phosphorylated histone H2A. Antibody X (Ser 139) (# 05636, Upstate Biotechnology, Inc., Lake Placid, NY, USA) was added. After washing, embryos were incubated with secondary antibody (Alexa dyes, Invitrogen, Carlsbad, CA, USA), washed again and observed.

[result]
Embryos created by in vitro fertilization (IVF) were observed under various conditions and transferred to pseudopregnant females (FIG. 2a). Highly sensitive green fluorescent protein (EGFP) (EGFP-α-tubulin) (Genesis 43, 71-79 (2005)) to which α-tubulin is bound, and monomeric red fluorescent protein (mRFP1) fused with histone H2B ( J Reprod Dev 53, 1035-1041 (2007)) was injected immediately after fertilization. The effects of mRNA concentration and fluorescence microscopy on embryo development are summarized in FIG. 3 and Tables 1 and 2.

^a mRNA was injected into late / terminal IVF embryos in the second meiosis, incubated for about 78 hours after insemination, and transplanted. A fluorescence photograph was taken immediately before embryo transfer and is shown in FIG.
^b Equal volume mixture of EGFP-α tubulin and H2B-mRFP1.
^c Compacted embryos with more than 8 nuclei were evaluated as morula / blastocyst stage.
^d Embryos were transferred to the uterus of pseudopregnant females 2.5 days after intercourse.

^a Injection of 25 ng / μl of each mixture of EGFP-α-tubulin and H2B-mRFP1 into IVF embryos and transfer to 5 μL CZB drops on glass bottom dish (12 embryos / drop) until blastocyst stage Incubated (about 70 hours). Two colors (green and red) of excitation light were irradiated at intervals displayed 51 times in the Z-axis direction.
^b Immediately prior to embryo transfer, embryos were observed once under a confocal microscope.
^c Compacted embryos with more than 8 nuclei were evaluated as morula / blastocyst stage.
^d Embryos were transferred to the uterus or oviduct of pseudopregnant females 2.5 or 0.5 days after intercourse.
^e The percentage of offspring was calculated as follows: (number of offspring) ÷ (number of morula / blastocyst embryo)

From the results shown in Table 1, by transplanting an embryo injected with mRNA at a concentration of 10 to 250 ng / μL into the uterus of a pseudopregnant female, a child derived from the embryo was born. At a concentration of 250 ng / μL, in vitro differentiation to the morula or blastocyst stage and the birth rate decreased, but at a concentration of 50 ng / μL, in vitro differentiation and a decrease in the birth rate were not observed.

From the results in Table 2, even when the embryo is exposed to excitation light with a laser power of 20-70 mW, the embryo differentiates in vitro to the morula or blastocyst stage and the embryo is transferred to the uterus of a pseudopregnant female As a result, offspring derived from the embryo were born. However, when fluorescence imaging was performed every 3.75 minutes with a laser output of 70 mW, the birth of the offspring was inhibited, but at an interval of 7.5 minutes, in vitro differentiation to the morula or blastocyst stage and birth rate There was no decline.

Surprisingly, a child was born even after the pre-implantation occurrence was tracked for a long time with a 6-dimensional video (FIGS. 2b, c and Table 2). These embryos were exposed to two different wavelengths of excitation light (488 and 561 nm) for approximately 70 hours at 7.5 minute intervals, yielding 51 images for the Z axis at each time point; , 814 fluorescence images were taken (Table 2). There was no difference in results when the probe combination was changed from EGFP-α-tubulin to EGFP-β-actin (Table 2). Since these pups grew to normal adults (FIG. 2d) and both males and females were normal in terms of fertility (data not shown), our imaging system is safe for embryonic development. It has been suggested. In addition, there was no transgenic out of 402 offspring because mRNA injection was used rather than DNA (Figure 2e, f and Table 2). This confirms the safety and efficiency of our PDI. The imaging device used (FIG. 2g) is shown in the method section and in FIG.

Most human babies born after ICSI are normal, but this ART has a lower pregnancy rate than those after natural conception (Reprod Biomed Online 10, 247-288 (2005); Hum Reprod 23, 756 -771 (2008)). Furthermore, chromosomal abnormalities were observed in mouse ICSI embryos (J Reprod Dev 53, 615-622 (2007); Biol Reprod 77, 336-342 (2007)).
We applied the PDI technique to assess the chromosomal integrity of mouse ICSI embryos. When first somatic division was monitored, cytokinesis was normal, but significant abnormalities were observed during chromosome segregation (FIGS. 5a-c). A small dot-like H2B-mRFP1 signal was detected along the microtubule bundle between late and late mitosis, which remained in the cytoplasm even in the stationary phase of the 2-cell embryo (FIG. 5b). Although there was no ectopic H2B-mRFP1 signal at the two-cell stage, another unusual pattern was also observed in which chromosome fragments were detached from metaphase plates and reattached during division (FIG. 5c). . In some embryos, a combination of the above two patterns was seen. In this study, all these abnormalities were classified as abnormal chromosome segregation (ACS) and the rest were collectively classified as normal chromosome segregation (NCS) (FIG. 5a). The frequency of ACS in ICSI embryos (10.4% -46.7%) was significantly higher compared to IVF embryos (1.7%) (Table 3).

^a mRNA was injected into the 2nd meiotic / end-stage embryo and 6-dimensional imaging was performed. Imaging was performed by the 2-cell stage, embryos were classified into their respective categories and transferred to pseudopregnant recipients.
^b FT, freeze thaw.
^c Operator W was a skilled micromanipulator and Y was a beginner at that time.
^d Embryos with 1 or 3 pronuclei (PN) were excluded from the analysis.
^e Embryos were classified into NCS and ACS based on the images in FIGS. 5a-c.
^f Two-cell embryos of each category were transplanted into the oviducts of pseudopregnant females 0.5 days after intercourse.
^{The g} value was calculated as the ratio of the total number of offspring to the total number of transplanted 2-cell embryos.
^h 65% of 2PN embryos had one large female pronucleus and one very small male pronucleus.
The frequency of ^{i, j} ACS was significantly higher than that after IVF ( ⁱ P <0.01, ^j P <0.05, Student's t-test) nd, untested.

Interestingly, the frequency of ACS in ICSI embryos was highly dependent on the operator's skill (Table 3). When freeze-thawed sperm were used for ICSI, the ACS percentage was significantly higher than when fresh sperm was used, even when the most skilled micromanipulators were operated (Table 3). Immunostaining analysis at the 2-cell stage revealed that chromosomal fragments suspended in the cytoplasm lacked laminB (FIG. 5d), but were positive for anti-phosphorylated histone H3 Ser10 antibody, a marker of aggregated chromosomes. (Figure 5e). This indicates that even though quiescent nuclei are formed in the 2-cell blastomeres, they are independently metaphase. In addition, these fragments contain anti-phosphorylated H2A. It was stained with X antibody (Biol Reprod 75, 673-680 (2006)) (Fig. 5f). These data suggest that ACS may result from cleavage of double-stranded genomic DNA, resulting in a chromosomal fragment that appears in metaphase and subsequently dissociating from the spindle microtubules.

When imaging the ICSI embryos to late stages, it was found that more than half of the ACS-bearing embryos differentiated into the morula / blastocyst stage (FIG. 6a and Table 4).

Create an ICSI embryos using sperm ^a freeze-thaw, mRNA was injected in phase anaphase / telophase of the second meiotic division, and imaged long time until the morula / blastocyst stage (about 70 hours) .
^b Compacted embryos with more than 8 nuclei were evaluated as morula / blastocyst stage. The rest were counted as “stopped” embryos.
^c Mulberry / blastocyst stage embryos were transplanted into the oviducts of pseudopregnant females 0.5 days after sexual intercourse. n. d. Means “untested”.

In some blastocysts, the pattern of cell lineage specification between trophectoderm (Cdx2 positive cells) and inner cell mass (Oct3 / 4 positive cells) was abnormal, but ACS embryos are Like the embryo, it had a blastocoel that looked normal (FIGS. 6c, d). To assess the ability of embryos with ACS to implant and post-implantation development, after live cell imaging, embryos were separated into ACS and NCS at the 2-cell stage and transferred separately to the oviducts of pseudopregnant females (Fig. 6e). Embryos with ACS could be implanted and formed a decidua (Fig. 6f), but no viable embryos disappeared at E7.5 and began to be absorbed at approximately E9.5 (Fig. 6g and Table 5). This suggests that embryos with ACS spontaneously aborted by E7.5.

Create an ICSI embryo using ^a freeze-thawed sperm to 2-cell stage for 24 hours imaging, sorted into ACS and NCS embryos manipulator and implanted into pseudopregnant females. Females were sacrificed after embryonic day 7.5 (E7.5) or 9.5 (E9.5) days, the reproductive organs were dissected and the uterus was removed.

In fact, in all ICSI experiments, no or very few offspring were obtained from ACS embryos, but NCS embryos developed normally into offspring (Tables 3 and 4). These data clearly show that the relatively low pregnancy rate after ICSI is due to abnormal chromosome segregation in the first somatic division followed by miscarriage before E7.5. Thus, PDI enables the identification of incomplete embryos from seemingly normal two-cell stage mouse embryos, and the overall pregnancy rate can be improved by removing such embryos from transplantation. did. We again emphasize that 2-cell embryos with ACS and 2-cell embryos with NCS cannot be distinguished at all by light microscopy (FIG. 7).

The present inventors investigated the prevalence of ACS in other ART models using PDI. Although round sperm cells are haploid like mature sperm, the successful birth rate after round sperm cell injection (ROSI) in mice is very low (1.7-28.2%) (Proc Natl Acad Sci USA 91) 7460-7462 (1994); Development 121, 2397-2405 (1995)). Imaging results show that many ROSI pronuclear stage embryos contain one very large female pronucleus and one very small male pronucleus (Figure 8a), which is consistent with previous reports ( Development 121, 2397-2405 (1995)). These embryos exhibited severe ACS in first somatic division (Fig. 8a) and none developed until the end (Table 3), but the birth rate of ROSI embryos with NCS was 44.4%. (Table 3). Since the birth rate after ROSI without PDI is estimated to be 10% (column “NCS + ACS” in Table 3), PDI has improved the pregnancy success rate by more than four times.

ROSI is an experimental model of low pregnancy, presumably due to sperm immaturity. The present inventors also examined chromosomal abnormalities derived from maternal factors. Chromokinesin Kid / kinesin-10 plays a key role in chromosome segregation by localizing at the boundaries of anaphase and metaphase chromosomes and mediating shortening of mitotic chromosome clusters along the spindle axis. (Mol Biol Cell 16, 5455-5463 (2005)). Kid ^{− / −} females are poorly fertile and are probably thought to be partly due to abnormal chromosome segregation of first somatic division (Cell 132, 771-782 (2008)). The present inventors performed PDI on embryos created by ICSI using Kid-/-oocytes. As reported in Cell 132, 771-782 (2008), many single-cell embryos have multiple pronuclei, after which the nuclear envelope has collapsed and all chromosomes have assembled into metaphase plates. Later, it normally split into two nuclei (FIG. 8b). However, 33.8% of these embryos showed ACS (FIG. 8b), almost all of which failed to develop after transplantation (Table 3), while 66.2% showed NCS, which 28.6% occurred to the end (Table 3).

(Example 2)
The same test as in Example 1 was performed using monkeys as models.
Microinsemination (ICSI) was performed on unfertilized eggs collected from cynomolgus monkey ovaries. As an unfertilized egg, an egg recovered from the ovary that was stopped in the second metaphase (metaphase II) was used. Sperm collected by electrical stimulation of the penis were suspended in the medium after centrifugal washing and incubated for 1 hour to acquire fertility, and only those that increased after centrifugation were used for microinsemination. After incubation for about 3 hours, EGFP-tubulin and Histone H2B-mRFP1 mRNAs adjusted to 5 ng / μl each were injected into embryos immediately before the pronuclear phase at about 20 to 30 picoliters per embryo.
After 3 hours of incubation, imaging was started from the pronuclear phase. For embryos that had two male and female pronuclei and showed normal cytokinesis during first somatic division, the dynamics of nuclei and chromosomes during the first somatic division were observed.

The conditions for live cell imaging of monkey embryos were the same as for mice except for the following points.
(1) The incubator temperature was set at 38 degrees.
(2) Observation was performed for about 60 hours from the 1-cell stage to around the 4-cell stage.
(3) Since the monkey embryo was larger in size than the mouse embryo, the imaging interval was changed from 2 μm to 3 μm of the mouse, and 46 images were acquired in the Z-axis direction.
(4) The shooting time interval was 15 minutes.

As a result, less than half (45.7%) showed normal chromosome segregation, and the rest all showed abnormal chromosome segregation. The types of abnormalities and their frequencies are shown in FIG. 9 and Table 6, respectively.

^aA male and female pronucleus with normal cytokinesis during the first somatic division.
^b Abnormalities commonly seen in mouse insemination embryos, in which part of the chromosome is misplaced during distribution (see FIG. 9A).
^c Abnormalities with one or more nuclei in either the 2-cell embryo or both blastomeres after division (see FIG. 9B).
^{d An} abnormality that causes chromosomes to become fragmented during division and to form innumerable nuclei in 2-cell embryos (see FIG. 9C).

Those that showed normal chromosome segregation were classified as NCS, and all other abnormal ones were classified as ACS, and each of them was further cultured for several days to examine the developmental ability to morula and blastocyst stage embryos (Table 7). Of those embryos that reached the blastocyst stage, embryo transfer was performed to examine the ability to produce offspring (Table 7). One or two blastocyst stage embryos were transferred to one foster parent.

^a Embryo whose development has stopped midway without reaching the morula.
^b Embryos with severe cytoplasmic fragmentation.

As a result, more than half of the NCS embryos developed into morula and blastocyst stage embryos, and a part of them was transplanted into the temporary parental oviduct. One of them was born due to spontaneous delivery at the time of pregnancy. On the other hand, about 10% of the ACS embryos were developed up to the morula and blastocyst stage, but no offspring were obtained as a result of the transplantation. From these results, it became clear that abnormal chromosome partitioning occurred frequently in the first somatic division in monkey microinseminated embryos as well as mice, which caused developmental abnormalities.

As described above, even in the case of using monkey embryos, as with mouse embryos, offspring can be obtained from the embryos after imaging, and by eliminating ACS embryos from transplantation, the pregnancy rate can be improved as a result. It was.

By using the method of the present invention, it is possible to observe the early embryo in detail while suppressing damage to the embryo as much as possible. By using the method of the present invention, it is possible to continuously observe fluorescence while keeping a mammalian embryo alive, and furthermore, the embryo can be individually generated without any problem. And the subsequent ontogeny can be directly linked and evaluated. Furthermore, by using the method of the present invention, it is possible to identify embryos at a very early stage, up to the 2-cell stage, at a high risk of miscarriage in the future. And it becomes possible to improve a pregnancy rate significantly by removing the identified abnormal embryo from the transplant object. In addition, in the method of the present invention, since the offspring born from the observed embryo is not transgenic, it can be applied to the medical / livestock field.
This application is based on Japanese Patent Application No. 2008-213296 filed in Japan (filing date: August 21, 2008), the contents of which are incorporated in full herein.

Claims (11)

1. A method for in vitro fluorescence observation of a mammalian embryo, comprising acquiring fluorescence images of a fluorescently labeled mammalian embryo over time with a confocal fluorescence microscope equipped with a laser light source and a nipou disc confocal unit.
2. The method according to claim 1, wherein the fluorescently labeled mammalian embryo is obtained by injecting RNA into the embryo that encodes the fluorescent protein and can be translated into the fluorescent protein in the embryo. .
3. The method according to claim 2, wherein the amount of RNA to be injected is a sufficient amount for obtaining a fluorescence image and not more than 2.5 pg per embryo.
4. The method according to claim 1, wherein fluorescence images of mammalian embryos are continuously acquired at a time interval of at least 3.75 minutes.
5. The method according to claim 1, wherein fluorescence images of mammalian embryos are acquired over time for at least 20 hours.
6. At the time of one observation, the focal point of the confocal fluorescence microscope is moved with respect to the embryo in the height direction with respect to the stage surface of the microscope as a reference at an interval of 3 μm or less, and the cross section of the embryo of each layer is The method of claim 1, wherein a fluorescent image is acquired.
7. A method for selecting a mammalian embryo with a low risk of implantation failure or miscarriage comprising the following steps:
   (I) preparing a mammalian embryo that expresses a fusion protein of a chromosome constituent protein and a fluorescent protein or a fusion protein of a spindle constituent protein and a fluorescent protein;
   (II) Obtaining a fluorescence image of the mammalian embryo obtained in (I) over time by a confocal fluorescence microscope equipped with a laser light source and a Niipou disc type confocal unit; and (III) obtained in (II) Based on the obtained fluorescence image, an embryo that does not exhibit abnormal chromosomal morphology is selected as a mammalian embryo having a low risk of implantation failure or miscarriage.
8. The mammalian embryo of step (I) encodes a fusion protein of a chromosome constituent protein and a fluorescent protein or a fusion protein of a spindle constituent protein and a fluorescent protein, and an RNA that can be translated into the fusion protein in the embryo 8. The method of claim 7, wherein the method is prepared by injection into the embryo.
9. 6. The method according to claim 5, wherein the mammalian embryo of step (I) is a 1-cell stage or 2-cell stage embryo.
10. The method according to claim 7, wherein the mammalian embryo is prepared by intracytoplasmic sperm injection (ICSI) or circular sperm cell injection (ROSI).
11. The method according to claim 7, wherein the abnormal chromosome morphology is abnormal chromosome segregation during somatic cell division.

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