WO2003008633A1

WO2003008633A1 - Screening method using fish embryo

Info

Publication number: WO2003008633A1
Application number: PCT/JP2002/007289
Authority: WO
Inventors: Yutaka Tamaru; Toshio Tanaka
Original assignee: Nagoya Industrial Science Research Institute
Priority date: 2001-07-18
Filing date: 2002-07-17
Publication date: 2003-01-30
Also published as: JP2003052354A

Abstract

It is intended to provide a method of screening a chemical which requires little labor and is usable as a substitute for vivisection and an apparatus therefor. A plate 4 made of a transparent material is provided with a plural number of wells 5. The wells 5 contain a liquid enabling the development of fish embryos and a test chemical. Fish embryos are soaked in the wells 5. After a definite period of time at a definite temperature, each embryo develops into an individual. In case where the test chemical affects the development of the embryo, the embryo is malformed or dies.

Description

Specification

Screening method using fish embryo

The present invention relates to a screening method using a fish embryo and the like. Background art

Now that the types of chemicals are enormous, it is very important to determine whether or not certain chemicals have hormone-like effects (endocrine disrupting properties). In the case of pharmaceuticals, examining the side effects in addition to the main effects of the pharmaceutical is an essential test item for providing the pharmaceutical to the factory.

As described above, in order to examine the effects of chemical substances on living organisms, such as mutagenic effects, carcinogenic effects, teratogenic effects, endocrine disrupting effects, etc., first, tests using cultured cells (in vitro tests) It is common to conduct a test using a living body (in vivo test) after conducting the test.

However, performing in vitro tests requires specialized equipment for maintaining cultured cells and assaying the effects, and requires significant human and economic labor. Also, in the in vivo test, a lot of labor is required for the maintenance of living organisms (some animals such as monkeys, butterflies, etc., in addition to small animals such as rats and mice) and for testing methods. Has become. In addition, ethical issues can arise with the use of animals as is.

As described above, the conventional methods of evaluating chemical substances have been extensive with much effort.

In order to solve the above-mentioned difficulties, evaluation techniques using fish have recently been disclosed (for example, WO99 / 42606, WO00 / 32882, etc.). ). However, this technology has not yet been fully developed and there are many points to be improved.

The present invention has been made in view of the above circumstances, and its purpose is to reduce labor. It is an object of the present invention to provide a method for screening chemical substances which can be used in place of a biological test and which can be used instead of a biological test. Disclosure of the invention

The present inventors have conducted intensive studies and found that, in the process of turning an animal from a fertilized egg into an adult, the stage of the embryo is most susceptible to chemicals. Furthermore, ② fish embryos are not only endocrine disruptors, It has been found that it is possible to completely expose any chemical substance by mixing it in a liquid, and that its effects can be detected with high sensitivity. Thus, the present inventors have basically completed the present invention.

The first invention for solving the above-mentioned problem is a plate provided with a well for holding a liquid to which a chemical substance can be added while a fish embryo is immersed, and a fish embryo inside the well of the plate can be observed. A screening device using a fish embryo, comprising an observation device and a photographing device capable of photographing an image obtained by the observation device.

In the following description, “in the present invention” means “in all the inventions described in this specification”.

There are no particular restrictions on the fish that can be used in the present invention, and teleosts' cartilaginous fish and jawless fish can be used.However, from the viewpoint of maintenance of test facilities, it is preferable to use fish that are as small and prolific as possible. . It is further preferred that the fish embryos are transparent. In addition, it is preferable to use a fish embryo having a short time (from several hours to several days) from the one-cell stage to becoming an individual. In order to compare the effects of chemicals on fish as individuals and the effects on humans, it is necessary to determine the genomic sequence of the fish or that it will be known in the near future. It is preferred to use Particularly preferred examples of such fish include trough, medaka, and zebrafish. In addition, it is preferable to select fish in which the number of eggs (embryos) obtained in one spawning is several tens or more. If the number of laid eggs is too small, it takes too much time to perform the evaluation test. It has been known that the sex of fishes fluctuates due to internal factors caused by genes and external factors caused by sex hormones and environmental conditions. So, like this A system for detecting environmental hormones such as endocrine disruptors has been developed using the sex determination process of fish and its mechanism. However, since these detection systems directly quantify sex hormones and their related substances in the blood of fish, the use of adult fish is based on seasonal variation and individual differences in fish due to age. The data varied widely. Also, when using small fish such as medaka, the difficulty of collecting blood was a major problem.

In the present invention, the term “fish embryo” means an egg in which a fish can develop as an individual, and an egg that is dividing in order to become an individual. In order to confirm the effects of chemicals on fish embryos, embryos should be as early as possible (before starting cleavage (1 cell stage) or immediately after cleavage (eg, 4 cell stage to 8 cell stage). )) Is preferred. This is because if the cleavage progresses to a certain extent, there is a concern that the action will not occur even if a chemical substance is added. It is most preferable to use a fish embryo before the start of cleavage. From another point of view, it is preferable to use one with high permeability of the egg membrane so that the chemical substance can easily enter the inside of the fish embryo. In addition, the egg membrane can be appropriately treated (for example, an enzyme treatment) in order to enhance the penetration of the chemical substance into the fish embryo. It is preferable to use a transparent fish embryo. By doing so, it becomes possible to observe organs in the embryo such as the spine and internal organs. An example of such a fish embryo is zebrafish.

In the present invention, “immersion” means that a fish embryo is immersed in a liquid in a state where cell division is possible until it becomes an individual. Normally, it is necessary to control the temperature to a constant state, but it is not necessary to control the partial pressure of oxygen, carbon dioxide, etc. in the air. Hassle-free. In the present invention, the term "chemical substance" is a general term for a substance which can be chemically synthesized, which can be isolated and purified from a natural product, or a mixture which is obtained naturally but whose components and component ratios are unknown. are doing. “Chemical substances” include, in addition to compounds of inorganic compounds and organic compounds, polypeptides and proteins (including those modified with sugar chains, etc.) that can be produced by genetic engineering. Of proteins and nucleic acid molecules, as well as nucleic acids (single, double, or triple stranded DNA or RNA). I will. In addition, it is preferable that the chemical substance has a property (water solubility) that dissolves in a liquid in which the fish embryo is immersed (in most cases, a liquid mainly composed of water (fresh water or seawater)). Even if it has no properties (or exhibits only a very small amount of water solubility), for example, a small amount of the solvent is added to the liquid in which the fish embryo is immersed, while being dissolved (or dispersed) in an organic solvent. This allows fish embryos and chemical substances to coexist. In the present invention, since the chemical substance acts directly on the fish embryo, the action of the chemical substance can be confirmed even at a relatively low concentration. In the present invention, "addition" means that the chemical substance to be tested is added in a liquid so that it can come into contact with the fish embryo. The chemical substance can be added before the fish embryo is immersed in the liquid, but may be added simultaneously with the fish embryo or after the fish embryo is immersed in the liquid. Is also good.

In the present invention, the term “liquid” is mainly composed of water in many cases. However, fresh water or seawater-like one containing salt at an appropriate concentration (including seawater itself) according to the fish used is often used. Can be used. In order to maintain the pH within a predetermined range, an appropriate buffer can be used.

In the present invention, “well” means a well-shaped depression that holds a liquid. It is preferable to use the minimum size of the wells according to the size of the fish embryo used. For example, when the initial size of the fish embryo is about 0.8 mm, a cylindrical well having a diameter of about 5 mm and a depth of about 5 mm can be used. In many cases, fish embryos sink into the bottom of the liquid in the wells. Therefore, (1) When observing fish embryos by illuminating the plate with transmitted light, it is preferable to have a flat bottom surface. This will make it easier to observe fish embryos by suppressing the reflection and scattering of light. In addition, (2) the position where the image is to be captured in the observation device can be determined, and a device with a mortar-shaped bottom can be used for the purpose of automation. This is because the fish embryo is fixed at the top of the mortar (the lowest point of the ゥ el).

In the present invention, the “plate” means a base portion having one or more wells. The material of the plate is not particularly limited, and it can be manufactured using resin (synthetic resin or natural resin) 'glass or metal. 、 From the viewpoints of operability and economy, it is preferable to use a synthetic resin. In such a case, it is more preferable that the plate is made of a transparent resin having high transparency. This makes it easier to illuminate one side of the plate and observe the fish embryo from the other side.

In the present invention, an “observation device” is a device for observing a state in which a fish embryo divides as an individual. If the division of the fish embryo is large enough to be observed with the naked eye, it is possible to use both the observation device and the imaging device.However, in many cases, the observation device is used to enlarge the fish embryo to be observed. It is preferable to have the ability to observe by observation. An optical microscope can be exemplified as the most suitable device for that purpose. However, if the photographing device has an enlargement capability, it can also be used as an observation device.

In the present invention, “shooting” means capturing an image obtained by an observation device, and in addition to a general photograph (including a black-and-white or a color photograph), analog data, or This includes storing images as digital data.

In the present invention, the “photographing device” means a device that can store an image obtained by an observation device. For example, a polaroid camera, a camera using a general developing film, a digital camera, a CCD camera, and an image recording device Devices (including floppy disks, CDs, portable recording media such as MOs, and hard disk-integrated image recording devices, etc.).

According to the first invention, a fish embryo and a chemical substance are added to the liquid in the well. Then, the changes in the fish embryo can be observed using an observation device, and the image can be taken by an imaging device when necessary, and the effect of the chemical substance on the development of the fish embryo can be evaluated through the photograph. .

In particular, fish embryos take less time to grow as individuals than mammals, so similar results can be obtained in a shorter time than conventional in vivo tests. In addition, fish embryos are easier to breed than mammals, and the whole test can be easily performed.

Also, by using fish embryos, compared to the case of using conventional cultured cell lines, ① Less labor for management (For example, the development of fish embryos does not require the management of components such as carbon dioxide in the atmosphere. Also, for small fish (for example, medaka and zebrafish, etc.), the labor for breeding is less. ), ② There are advantages such as a short time from the early embryo to the individual. In addition, (3) the use of fish embryos for which genome sequence data is available (eg, troughfish, medaka, zebrafish) allows the use of chemical substances between mammals (especially humans) and fish. It will be possible to correlate the effects on genes.

A second invention is characterized in that, in the first invention, the plate is formed of a transparent material, and the bottom surface of the well is flat.

According to the second invention, when observing a fish embryo in a well, light is illuminated from one side of the plate if necessary (if the surroundings are sufficiently bright, it is not necessary to illuminate the fish). No), observation by the observation device and imaging by the imaging device are performed from the other side. This makes it easier to observe the fish embryo than a reflective plate (a type in which the bottom of the well is configured to reflect), since the image of the fish embryo is not duplicated. The observation by the observation device and the imaging by the imaging device may be performed on either the upper surface or the lower surface of the plate. Furthermore, when the number of wells on the plate is small, observation and photographing can be performed from the side of the plate.

The third invention is a screening method using a fish embryo, which is characterized by immersing a fish embryo in a liquid containing a chemical substance and observing a change over time of the fish embryo. According to the third aspect, since the chemical substance is contained in the liquid, it can be in contact with almost the entire fish embryo, so that the effect of the chemical substance can be reliably obtained. . In addition, by using fish embryos at the very early stage (for example, from the 1-cell stage to the 8-cell stage), it is necessary to confirm the effects that chemical substances can have at all stages from the initial stage of development to the individual stage Can be. At this time, it is preferable to remove the egg membrane of the fish embryo. As a method for removing the egg membrane, a mechanical treatment or a chemical treatment can be used, but it is preferable to perform a chemical treatment using an enzyme (for example, protease E can be used). Further, it is preferable that the removal treatment of the egg membrane is performed when the egg membrane has two layers.

In the invention of the method, fish embryos are the first point in time when a chemical substance is contained in a liquid. A point in the phasing stage (1 cell stage to 8 cell stage) is preferred. However, if it is necessary to select the point of addition of the chemical, such as when it is desired to evaluate at which stage of the embryo the chemical will cause the most damage, Thus, an appropriate point of addition can be selected.

A fourth invention is characterized in that, in the third invention, a time-lapse observation point is at least between a time point when organ formation starts and a time point when individual formation is completed.

In order to observe the effects of chemicals on fish embryos, it is necessary to start at least the time of organ formation (the time at which the fish embryo begins to form organs such as the spine, eyes, and internal organs)-the time of completion of individual formation (at It is important that these points are between the two points (the point at which each organ can be moved by the caudal fin). The reason is that if the action of chemicals is strong, organ formation may be inhibited at the former time point, and even if the action of chemicals is weak, malformation will eventually occur in an individual Because it appears. Specifically, for example, in the case of zebrafish, organ formation starts at about 26 ° C at a water temperature of about 12 hours after the 1-cell stage, and when the About 24 to about 30 hours after the 1-cell stage. Therefore, in the case of zebrafish, it is possible to confirm the effect of the chemical substance on the fish embryo by performing observations from about 12 hours to about 30 hours after the 1-cell stage. is there.

Further, a fifth invention is a pharmaceutical composition characterized by being screened in the third invention or the fourth invention (invention of a method). According to the method of the present invention, the effect of a chemical substance on a fish embryo (living body) can be easily evaluated, so that the method can be applied to a pharmaceutical composition. In the fifth invention, the “pharmaceutical composition” means a substance applied to a pharmaceutical among the “chemical substances” in the present invention.

According to the present invention, the action of a chemical substance can be confirmed using a fish embryo. The process of fish embryo development can be imaged over time with an imaging device. Large-scale screening of chemical substances is possible according to the number of spawned fish, thus enabling high-throughput data. In addition, the development time of fish embryos is much shorter than that of conventional in vivo tests, so that the time can be shortened. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram showing a state when a fish embryo is being processed in the present embodiment.

FIG. 2 is a photograph of the plate in the present embodiment.

FIG. 3 is a view showing a state in which fish embryos and chemical substances are dispensed into the wells of the plate in the present embodiment.

FIG. 4 is a photograph showing a state in which a digital camera is mounted on the eyepiece side of the microscope in the present embodiment and the state of the fish embryo in the well is observed.

Fig. 5 is a photograph showing the appearance of zebrafish embryos when no chemical substance is added.

(A) shows an embryo at 0 hours, (B) shows an embryo at 12 hours, and (C) shows an embryo at 24 hours.

FIG. 6 is a photograph showing a state of development of zebrafish embryos when 25 g / m 1 of magnesium sulfate was added.

FIG. 7 is a photographic diagram depicting the appearance of zebrafish embryos when 100 μg / m 1 of magnesium sulfate was added.

(A) shows an embryo at 0 hours, (B) shows an embryo at 12 hours, and (C) shows an embryo at 24 hours. BEST MODE FOR CARRYING OUT THE INVENTION

Next, an embodiment of the present invention will be described in detail with reference to the drawings. However, the technical scope of the present invention is not limited by the following embodiment, and the gist thereof is not changed. However, various modifications can be made. The technical scope of the present invention extends to an equivalent range.

Prepare a dilution of the chemical substance at an appropriate magnification, and place a fish embryo in each well of the plate. Dispense with the liquid according to.

Fish containing male and female pairs are kept in cases. Transfer a good pair to a netted case and wait for spawning. After confirming the spawning, take out the net, transfer the contents of the net into a petri dish, and remove food, feces and immature eggs. Preferably, the following activities should be started within the shortest possible time (eg, within one hour) after laying eggs. If the time lapse from the spawning is too long, the cleavage will progress and the sensitivity to chemicals will decrease.

Transfer eggs to a suitable beaker, wash 2-3 times with the specified liquid, and pour an appropriate amount of liquid. <Egg membrane treatment>

The egg membrane is treated to soften the egg membrane and promote the penetration of chemicals into the fish embryo. Add a solution containing the appropriate protein-digesting enzyme to the liquid containing the eggs (fish embryos) and mix gently. Observe the eggs under a microscope for about 5 to 10 minutes until the egg membrane has two layers. When the egg membrane has two layers, gently add liquid to break the egg membrane, and then discard the liquid. Repeat the addition and removal of liquid until the broken egg membrane is removed.

Prepare an appropriate number of liquids in a Petri dish (prepared according to the type of test chemical), and dispense fish embryos treated with protein digestive enzymes into the Petri dish. Fig. 1 shows the situation at that time. In the figure, reference numeral 1 denotes a petri dish, and reference numeral 2 denotes a pipette. Reference numeral 3 is a fish embryo.

Next, one fish embryo 3 is transferred to each well 5 of the plate 4 as shown in FIG. Figure 3 shows the situation at this time. FIG. 3 shows a state in which a plurality of plates 4 are stacked and sequentially processed.

Observation of fish embryo>

Immediately after transferring the fish embryo, observe the state of the fish embryo with a microscope. At this time, if the fish embryo has already ruptured, prepare another fish embryo as a result of physical shock or the like (not as an effect of a chemical substance). It is preferable to prepare multiple (preferably about 10) fish embryos for one concentration of one chemical substance. Each plate is placed inside an incubator that has been previously set to the temperature appropriate for the fish embryo. When the specified observation point has been reached, remove the plate and photograph the fish embryos in the well with an imaging device. FIG. 4 shows a state in which the plate 4 is placed on the microscope 6, each gel 5 is observed, and an image is taken by the digital camera 7 as an imaging device. Example

Next, an example in which chemical substances were evaluated using zebrafish embryos will be described. The zebrafish embryo becomes an individual approximately 24 hours after the start of the test, and the eyes, notochord, blood vessels along the notochord, and viscera can be identified. In addition, zebrafish embryos are small (approximately 0.8 mm in diameter) and highly transparent, making them suitable for microscopic observation.

For the evaluation device, a plate with 384 wells, a microscope capable of observing fish embryos inside the well, and a photographing device capable of taking a microscope image were used. More specifically, the plate is made of transparent plastic so that transmission type observation with a microscope is possible, and the well is a cylindrical one with a diameter of about 5 mm and a depth of about 5 mm. Was. The bottom of the well is flat. The microscope used was an inverted microscope made by NI KON, and the imaging device used was a digital video camera (NI KON COOL PIX Microsystem IV) and a digital camera (NI KON COOL PIX E990).

Zebrafish were bred by a small fish breeding system (MS water tank manufactured by Meito Suizou Co., Ltd.).

Plate processing>

Magnesium sulfate was selected as the chemical. It is said that administration of magnesium sulfate causes hypermagnesemia and may cause magnesium poisoning (low blood pressure, central nervous system depression, cardiac function depression, respiratory paralysis, etc.).

HS buffer _{(CaCl 2 0. lg, NaCl 3.5g} , KC1 0.05g, dissolved in purified water NaHCO ₃ 0.2 g To 1 liter), and prepare magnesium sulfate diluted to a concentration of 0 μg / 1, 25 μg / m1, and 100 μg / m1. Was dispensed at 35 μl. The 384-hole plate is provided in 16 columns and 24 rows. Since 10 fish embryos were used for one concentration of one chemical, one horizontal row (24 ゥ) was assigned for one concentration of one chemical. The rightmost column (16-well) was used as a negative control (only S buffer without chemicals), and the remaining bottle was used for additional use (fish embryos ruptured in the early stage). Preliminary case).

One male and female zebrafish was transferred to a netted case (a minimum of five cases were prepared per thirteen). When the female laying eggs was confirmed, the processing was performed as in the item of <Egg laying> in the above embodiment. Zebrafish produce about 200 eggs per egg.

The eggs were transferred to a 50 ml beaker, washed 2-3 times with HS buffer, and then poured into about 10 ml of HS buffer.

In an HS buffer containing eggs (fish embryos), 5 to 10 drops of 30 mg / ml Protease E were added and mixed gently. Observe the egg with a microscope until the egg membrane becomes two layers (about 5 to 10 minutes). When the egg membrane becomes two layers, gently add HS buffer to destroy the egg membrane, and remove the HS buffer. The liquid was discarded. The addition and removal of the HS buffer was repeated until the broken egg membrane was removed. In addition, zebrafish is easy to process because the egg membrane is soft.

Petri dishes were prepared by adding 1 to the number of test chemicals, and HS buffer was poured appropriately. The zebrafish embryos treated with ProteaseE were dispensed into the petri dishes. The embryos were then transferred one by one into each well of the plate (in which the specified concentration of chemicals and HS buffer were poured).

Observation of zebrafish embryos>

Immediately after transferring the zebrafish embryo, the state of the embryo was observed under a microscope. At this time, If the embryo was already ruptured, another embryo was prepared, as if it were due to physical shock (not due to the effects of chemicals).

Each plate was placed inside an incubator set at about 26 ° C (this time was set to 0 hour). Embryos were photographed with a digital camera at 0, 3, 6, 12, and 24 hours. The 0, 6, and 12 hours shooting was performed at a magnification of 100x (eyepiece x 10x, objective lens x 10x), and the 24th shooting was 40x (eyepiece). (X 10x, objective lens X 4x).

The results of the above test are shown in Table 1 and FIGS.

Evaluation: abnormal-0 1 2 3 4 5-> normal

FIG. 5 shows microscopic views of zebrafish embryos at 0, 12, and 24 hours in the control (magnesium sulfate, Og / ml). At 0 hour (Fig. 5 (A)), the embryo was almost spherical, and a line dividing the embryo into two was observed near the center. At 12 hours (Fig. 5 (B)), fish embryo development has progressed to a considerable extent, with the individual curving around the entire perimeter of the yolk. In addition, the occurrence of eyes, notochord and internal organs was confirmed. At 24 hours (Fig. 5 (C)), the formation of the individual is almost complete, although the yolk remains. From the head to the front of the abdomen, the zebrafish individuals should be smoothly curved approximately in the shape of a hemisphere along the yolk, and from the center of the abdomen to the tail, they should extend almost linearly along the notochord. Was observed.

Figure 6 shows that the sample was immersed in HS buffer containing 25 μg Zm1 of magnesium sulfate. The appearance of the zebrafish embryos at 0, 12, and 24 hours was shown. At time 0 (Fig. 6 (A)), the morphology of the embryos was similar to the control. On the other hand, at the 12th hour (Fig. 6 (B)), the development of the fish embryo progressed, and although the individual was curved along the outer periphery of the yolk, it was observed only up to about half a circle, By comparison, their size was clearly smaller. In addition, no eye formation was observed and chordal development was insufficient. No viscera was formed. 24th hour (fig.

6 (C)), although the formation of the individual was considered to be almost complete, no development of the eyes and head was observed, the notochord was bent, and a relatively large malformation occurred as a whole. .

FIG. 7 shows the state of the zebrafish embryos immersed in HS buffer containing 100 μg / m 1 of magnesium sulfate at 0, 12, and 24 hours. At 0 hours (FIG. 7 (A)), the morphology of the embryo was almost the same as that of the control (however, no line across the embryo was observed depending on the orientation of the embryo). In addition, 12 hours (Figure

In 7 (B)), the development of fish embryos progressed to a considerable extent, and the development of eyes, notochord, and internal organs was confirmed. In addition, the individual grew while bending along almost the entire circumference of the yolk, and the growth was almost equal to that of the control. However, at 24 hours (Fig. 7 (C)), no individual was observed, and the entire embryo became a gourd-shaped and was darkened entirely. This is because embryo development stopped halfway and died.

Table 1 shows the above results in numerical form. The same form as that of the control was regarded as normal, and the results were shown in five stages (normal value was set to 5 and death was set to 0). Magnesium sulfate at 25 μg Zm 1 showed values of 5, 3, and 2 over time (0, 12, and 24 hours). In addition, with respect to magnesium sulfate of 100 / Xg / m1, numerical values 5, 5, and 0 were shown over time.

Claims

The scope of the claims

1. A plate equipped with a well for holding a liquid to which a chemical substance can be added while the fish embryo is immersed, an observation device capable of observing the fish embryo inside the well of this plate, and an image obtained by this observation device A screening device using a fish embryo, comprising a photographing device capable of photographing.

2. The screening device according to claim 1, wherein the plate is formed of a transparent material, and a bottom surface of the well is formed in a flat plate shape.

3. A screening method using fish embryos, characterized by immersing fish embryos in a liquid containing chemical substances and observing the changes over time of the fish embryos.

4. The screening method according to claim 3, wherein the time-lapse observation point is at least between a time point at which organ formation starts and a time point at which individual formation is completed.

5. The screening method according to claim 3, wherein the fish embryo is transparent to the extent that an internal organ is observable.

6. The method according to any one of claims 3 to 5, wherein the fish embryo is one of a group consisting of trough, medaka and zebrafish.

7. The screening method according to any one of claims 3 to 6, wherein the fish embryo is a zebrafish embryo.

8. The screening method according to claim 7, wherein the time-lapse observation point is included within about 12 hours to about 30 hours after the 1-cell stage.

9. A pharmaceutical composition which is screened by the method according to any one of claims 3 to 8.

10. The egg membrane is removed from the zebrafish embryo, and a liquid containing a chemical substance and the embryo, from which the egg membrane has been removed, are immersed in a well of a plate made of a transparent material, and the embryo has one cell. A screening method, wherein the embryo is observed by light transmitted through the plate from about 12 hours to about 30 hours after the stage.

11. The screening method according to claim 10, wherein protease E is used to remove the embryonic egg membrane.

1 2. When the egg membrane of the embryo becomes two layers, the egg membrane is removed. 12. The screening method according to claim 10 or claim 11.

13. The screening method according to any one of claims 10 to 12, wherein the well of the plate has a flat bottom shape or a mortar shape.

14. The screening method according to any one of claims 10 to 13, wherein the plate is kept at about 26 ° C during the observation of the embryo.

15. The screening method according to any one of claims 10 to 14, wherein the plate is provided with 384 wells.