WO2015152330A1 - Système de reconstitution acellulaire utilisant un extrait d'ovocytes de mammifères - Google Patents

Système de reconstitution acellulaire utilisant un extrait d'ovocytes de mammifères Download PDF

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WO2015152330A1
WO2015152330A1 PCT/JP2015/060344 JP2015060344W WO2015152330A1 WO 2015152330 A1 WO2015152330 A1 WO 2015152330A1 JP 2015060344 W JP2015060344 W JP 2015060344W WO 2015152330 A1 WO2015152330 A1 WO 2015152330A1
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chromatin
sperm
mixture
fraction
egg
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PCT/JP2015/060344
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Japanese (ja)
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白髭克彦
大杉美穂
井上玄志
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国立大学法人東京大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0608—Germ cells
    • C12N5/061—Sperm cells, spermatogonia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/04—Coculture with; Conditioned medium produced by germ cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2503/00—Use of cells in diagnostics

Definitions

  • the present invention relates to a cell-free reconstitution system using an egg extract derived from a mammal.
  • sperm nuclear chromatin is highly condensed by protamine, protamine is replaced by egg cell-derived histones, and then various proteins and transcription factors bind to the genome, resulting in extensive reconstruction of sperm nuclear chromatin. Is done. So far, as a method to reproduce the reconstitution of sperm nuclear chromatin after fertilization in vitro, a technique using a cell-free system using an egg extract prepared from Xenopus eggs has been developed. .
  • Non-Patent Documents 1 to 3 Non-Patent Documents 1 to 3
  • an object of the present invention is to provide a method for efficiently inducing a chromatin such as sperm nuclear chromatin to the pronucleus using a mammalian egg extract.
  • the egg After removing the transparent body of the unfertilized egg obtained from the mouse and activating it, the egg is crushed and centrifuged (centrifugation; 3,800 g, 20 minutes, 4 ° C.) to clear the layer next to the fat layer It is separated into a fraction, an opaque fraction in the lower layer, and a female chromosome in the lowermost layer.
  • the inventors treated the sperm nuclei permeabilized with the transparent fraction and then processed the transparent fraction and the opaque fraction as the second treatment, and about 50% of the sperm nuclei were pronuclear. It was confirmed that it was induced to.
  • the inventors further added an opaque fraction or a mixture of an opaque fraction and a transparent fraction to the sample after the second treatment as the third treatment, and the induction rate of the pronuclei was about We found that it rose to around 60%.
  • the present invention includes the following (1) to (5).
  • a method for inducing a pronucleus from chromatin comprising the following steps (a) to (c): (A) a first step of mixing chromatin and a transparent fraction of an egg extract prepared from a mammal-derived activated egg, and incubating the mixture; (B) a second step of adding an egg extract or an opaque fraction to the mixture after the first step and incubating the mixture; (C) Third step of adding egg extract or opaque fraction to the mixture after the second step, incubating the mixture, and confirming whether chromatin in the mixture has differentiated into pronuclei ( 2) The method according to (1) above, wherein the chromatin is sperm nuclear chromatin.
  • a method for examining the fertilization ability of mammal-derived sperm which comprises the following steps (a) to (c): (A) a first step of mixing a sperm permeabilized with a sperm cell membrane and a transparent fraction of an egg extract prepared from a mammal-derived activated egg, and incubating the mixture; (B) a second step of adding an egg extract or an opaque fraction to the mixture after the first step and incubating the mixture; (C) Third step of adding egg extract or opaque fraction to the mixture after the second step, incubating the mixture, and confirming whether sperm in the mixture has differentiated into pronuclei ( 4) The method according to any one of (1) to (3) above, wherein the incubation time of the first step and / or the second step is 0 to 90 minutes. (5) A kit for performing the method according to any one of (1) to (4) above, comprising an instrument for preparing an egg extract.
  • the present invention it has become possible to efficiently induce the pronucleus from chromatin, for example, from sperm nucleus chromatin. As a result, for example, the fertilization ability of sperm such as livestock can be examined in advance.
  • FIG. 1 The result of having observed the process of differentiation of the sperm nucleus chromatin at the time of performing to a 2nd process is shown. In a, the processing flow up to the second step is schematically shown.
  • b is the process of differentiation of sperm nuclear chromatin when the treatment up to the second step is performed (decondensation, assembly of nuclear membrane precursor vesicles on the chromatin surface, fusion of nuclear membrane precursor vesicles, pronuclear Induction rate of formation and enlargement of pronuclei) was shown.
  • the result of having observed the process of differentiation of the sperm nucleus chromatin at the time of performing to a 3rd process is shown.
  • the flow of processing up to the third step is schematically shown in a.
  • b is the process of differentiation of sperm nuclear chromatin when the treatment up to the third step is performed (decondensation, assembly of nuclear membrane precursor vesicles on the chromatin surface, fusion of nuclear membrane precursor vesicles, pronuclear Induction rate of formation and enlargement of pronuclei) was shown. It is the result of examining the structural characteristics of the pronucleus formed by the method of the present invention.
  • a is a photomicrograph of the male pronucleus induced by the method of the present invention.
  • b is the result of examining whether dextran having a molecular weight of 70,000 and 10,000 molecular weight of Texas Red conjugate enters the pronucleus.
  • c shows the result of staining the pronucleus with Hoechst 33342 (DNA), anti-lamin A / C antibody (lamin A / C) and anti-nucleoporin antibody (mAb414).
  • Scale bar is 20 ⁇ m.
  • the first aspect of the present invention is a method for deriving a pronucleus from chromatin comprising the following steps (a) to (c).
  • chromatin and a mammal-derived unfertilized egg are prepared.
  • the mammal is not particularly limited, for example, animals used for experiments such as mice, rats, guinea pigs, rabbits, domestic animals such as cows, horses, sheep, goats, pigs, etc., dogs,
  • the concept encompasses all mammals such as cats, primates and humans.
  • chromatin is a complex of DNA and protein present in eukaryotic cells. Chromatin usually has a nucleosome structure in which DNA is wrapped around basic proteins such as histones, but chromatin in the sperm nucleus consists of protamine and DNA, not histones.
  • the chromatin referred to in the present specification is not limited to chromosomal DNA, and any DNA may be included, and the protein constituting the chromatin is histone, protamine, or other basicity.
  • the protein is not particularly limited as long as it is a protein that constructs a nucleosome structure.
  • Examples of the chromatin used in the present invention include sperm nuclear chromatin that has permeabilized the sperm cell membrane.
  • the present invention is also a method for inducing chromatin to the pronucleus.
  • the pronucleus is a nucleus in the previous stage where a male-derived chromosome and a female-derived chromosome merge at the time of fertilization.
  • a sperm-derived pronucleus is a male pronucleus and an egg cell-derived pronucleus is a female pronucleus. Called.
  • the pronucleus is usually a haploid nucleus. However, if the male and female chromosomes are very close to each other before the formation of the nuclear membrane, a double pronucleus including both is formed, which may also be called the pronucleus.
  • DNA contained in the pronucleus is chromosomal DNA derived from male or female, but in the present invention, the contained DNA is not limited to chromosomal DNA as long as it has the characteristics of the pronucleus.
  • Egg collection of unfertilized eggs can be performed by a method suitable for each animal species. For example, serum from gonadotropin (PMSG) or chorionic gonadotropin (CG) is injected into the target mammal to artificially induce an over-ovulatory state, and unfertilized eggs are collected. can do.
  • the dosage of these hormonal agents can be appropriately selected by those skilled in the art according to the type of mammal, body weight, and the like.
  • the collected unfertilized egg is used after removing the transparent body. Of course, it may be an unfertilized egg that has been naturally ovulated.
  • a well-known method in the technical field can be used. For example, in addition to a method using a laser or a method of physically peeling using a fine glass tube, acidic tyrode or SH group is removed. Untransparent eggs can be treated with a reducing agent (such as DTT) or the like to remove the transparent body. Next, the unfertilized egg from which the transparent body has been removed is artificially activated. Activation is a process for artificially reproducing the situation in an egg cell caused by sperm fertilization of an unfertilized egg in vivo.
  • a reducing agent such as DTT
  • Methods for artificially activating unfertilized eggs include, for example, methods such as calcium ionophore, ethanol treatment and electrical stimulation to increase the cytoplasmic calcium concentration of eggs, as well as 6-dimethylaminopurine, puromycin, cycloheximide, etc. And a method for inhibiting the synthesis of MPF (maturation-promoting factor).
  • an egg is physically pulverized, and an egg after pulverization is obtained.
  • Examples thereof include a method for preparing an egg extract by centrifugation.
  • the upper layer is divided into four fractions: a fat fraction, a transparent fraction, an opaque fraction, and a fraction containing female chromosomes. Centrifugation conditions should be adjusted so that they are separated in minutes.
  • the opaque fraction has a higher viscosity than the transparent fraction. Centrifugation conditions vary depending on the amount of eggs used and the centrifuge used, but those skilled in the art can easily determine by conducting preliminary experiments in advance.
  • a four-layer fraction containing a fat fraction it may be centrifuged using a normal centrifuge tube.
  • the unfertilized egg of a mammal is very small and the amount collected is small. Because there are many, a normal centrifuge tube is too large, and it may be difficult to prepare each fraction. In such a case, in order to prepare each fraction efficiently, for example, as devised by the inventors, a fine glass whose tip is closed with an appropriate adapter in a centrifuge tube used for normal centrifugation. It is possible to prepare four-layer fractions by fixing the tube in a centrifuge tube, filling the glass tube with eggs, crushing the eggs in the glass tube, and then centrifuging.
  • One side of the hematocrit tube can be closed with a putty for sealing, etc., and after filling the eggs, it can be fractionated by a centrifuge for hematocrit tube.
  • a centrifuge for hematocrit tube As described above, an unfertilized egg derived from a mammal with few fat granules can be separated into three fractions by centrifugation. As described in FIG. 1, when an egg is crushed and centrifuged using a glass tube, it is separated into three fractions within the glass tube.
  • the lowermost layer is a fraction containing female chromosomes
  • the intermediate layer is an opaque fraction (referred to as an opaque fraction)
  • the uppermost layer is a transparent fraction (referred to as a transparent fraction).
  • the transparent fraction is separated into four fractions in which a fat layer is formed in the upper layer.
  • the “egg extract” basically refers to a mixture of a transparent fraction and an opaque fraction. If the usage is different from this definition, an explanation will be given.
  • the temperature is preferably low, for example, about 4 ° C. It is desirable to prepare the transparent fraction and the opaque fraction immediately before use, but in some cases, a pre-prepared one can be stored at 4 ° C. and used within 2 hours after the preparation.
  • the chromatin used in the present invention may be prepared from mammalian cells, or any chromatin prepared by an appropriate method may be used (a method for preparing chromatin from any DNA is, for example, Xenopus egg As a method using an extract, Glikin GC. 1984, Cell. 37: 33-41, as a method using a Drosophila embryo extract, Ito T. 1997, Cell. 90: 145-155, 1997, and chromatin forming factor ACF Examples of the method include those described in Ito T. 1999, Genes & Dev. 13: 1529-1539).
  • a permeabilized mammalian sperm cell membrane can be used as the chromatin used in the present invention.
  • Mammalian sperm can be recovered from ejaculate or epididymis (see, eg, Fraser LR, 1984 J. Reprom. Fertil. 72: 373-384, 1984).
  • the collected sperm is washed with an appropriate buffer or the like (for example, PBS or the like), then the head and tail are separated, and the head is used.
  • the separation of the head and tail is not particularly limited, but can be performed by ultrasonic treatment or the like.
  • the sperm cell membrane is treated with a reducing agent such as DTT to make it permeabilized (see FIG. 1).
  • Sperm that has permeabilized the sperm cell membrane can be stored at 4 ° C. until use, but it is desirable to use it within 24 hours. Further, it can be stored frozen at -20 ° C or -80 ° C, but it is preferably used within one week.
  • sperm nucleus chromatin is added to the prepared transparent fraction and incubated. In this case, it is added so that the ratio of one sperm nucleus chromatin per egg cell, that is, the ratio of one sperm nucleus chromatin per transparent fraction of about 200 pl. It is desirable to appropriately treat the transparent fraction so as not to dry during the incubation.
  • a process in which the transparent fraction is in contact with the atmosphere may be covered with oil (mineral oil or the like).
  • the transparent fraction to which sperm nuclear chromatin has been added is incubated, for example, at 35 ° C. to 39 ° C., preferably at 37 ° C., in a gas phase of about 5% CO 2 (first step).
  • the incubation time in the first step is about 0 to 90 minutes, preferably about 30 minutes.
  • the egg extract (transparent fraction + opaque fraction) or the opaque fraction is added to the mixture after the first step and incubated (second step).
  • the amount of egg extract or opaque fraction added in the second step is about 6 to 6 times, preferably about 4 times the amount of the transparent fraction in the first step (about 800 pl).
  • Incubation in the second step is performed, for example, under a gas phase of about 5% CO 2 at 35 ° C. to 39 ° C., preferably at 37 ° C., for about 0 to 90 minutes, preferably about 30 minutes.
  • the egg extract or opaque fraction added in the second step is preferably newly prepared.
  • an egg extract (transparent fraction + opaque fraction) or an opaque fraction is further added to the mixture after the second step and incubated (third step).
  • the amount of egg extract or opaque fraction added in the third step is about 3 to 3 times, preferably about 2 times the amount of the transparent fraction in the first step (about 400 pl).
  • Incubation in the third step is carried out, for example, in a gas phase of about 5% CO 2 at 35 ° C. to 39 ° C., preferably 37 ° C., for about 0 to 90 minutes, preferably about 30 minutes.
  • the egg extract or opaque fraction added in the third step is preferably newly prepared. After the third step of incubation, pronuclei are formed.
  • Whether or not a pronucleus has been formed can be determined based on whether or not it has the characteristics of a nucleus. For example, confirm that functional nuclear pores containing nucleoporins are formed, the nuclear membrane lining protein, for example, lamin A / C, and encapsulated chromatin are decondensed. It can be judged by doing.
  • the second aspect of the present invention is a method for examining the fertilization ability of mammal-derived sperm, which comprises the following steps (a) to (c).
  • C Third step of adding egg extract or opaque fraction to the mixture after the second step, incubating the mixture, and confirming whether sperm in the mixture has differentiated into pronuclei When breeding animals, it is important to use sperm with fertilizing ability.
  • the present invention provides a method for examining in advance whether sperm from a mammal is induced to the pronucleus. Specifically, as the first form of chromatin, sperm with sperm cell membrane permeabilization and sperm nuclear chromatin are used to carry out the steps (a) to (c) above, and the induction rate to the pronucleus Confirm.
  • the induction rate to the pronuclei is good or bad, for example, using sperm that has already been clearly shown to have good induction in the pronucleus as a positive control and comparing it to the induction rate of that sperm Can be performed.
  • the third aspect of the present invention is a kit used for carrying out a method for inducing a pronucleus from chromatin and a method for examining the fertilization ability of mammal-derived sperm.
  • various hormones eg, PMSG, CG, etc.
  • reagents for permeabilizing the sperm cell membrane eg, streptricin O, DTT, etc.
  • Reagents for removing transparent bodies of unfertilized eggs for example, reducing agents such as acidic Tyrode and DTT
  • reagents for activation of unfertilized eggs for example, calcium ionophore, 6-dimethylaminopurine, puromycin, etc.
  • the kit of the present invention includes an instrument for preparing an egg extract, for example, an adapter used for centrifugation for preparing an egg extract, and a glass tube for preparing an extract from a small amount of eggs. You may go out.
  • an adapter for centrifugation for example, mold a glass tube using thermosetting resin or thermoreversible resin and cure it by applying heat, or mold it with silicon, and then pour and cure the liquid resin. It can be created by cutting out from metal and polishing.
  • the glass tube to be used can be prepared, for example, by narrowing the tip of a fine glass tube such as a hematocrit tube with a puller and then closing the tip with a microforge.
  • a buffer such as PBS
  • mineral oil for preventing drying during the incubation of chromatin and egg extract, etc. may also be included in the kit of the present invention. .
  • FIG. 1 The outline of the present invention is shown in FIG. 1 using as an example the case of using a sperm nucleus chromatin permeabilized with the cell membrane of mouse-derived sperm as the chromatin of the present invention and using an egg extract prepared from an unfertilized mouse egg Indicated. This will be specifically described below.
  • a parallel wire electrode chamber with a width of 0.5 mm is used, the space between the electrodes is filled with M2 medium (SIGMA), and the egg cell is placed in the center between the electrodes, and electrical stimulation at 200 V / mm, 99 ⁇ sec is performed for 0.5 seconds. Added four times at intervals (BEX LF101, Becks). After electrical stimulation, it was allowed to stand for 5 minutes in an M2 solution containing cytochalasin B (5 ⁇ g / mL).
  • the tip of the glass tube pulled with a puller was processed with a microforge and packed into a closed fine glass tube, and ATP regenerating system (1 mM ATP, 20 mM creatine phosphate, 20 U / mL creatine kinase, 1 mM GTP)
  • ATP regenerating system (1 mM ATP, 20 mM creatine phosphate, 20 U / mL creatine kinase, 1 mM GTP)
  • the glass tube was loaded into a 1.5 mL Eppendorf tube equipped with a handmade adapter, and lightly centrifuged (2,000 g / 15 sec / room temperature) to pellet the egg cells.
  • the centrifuged egg cell fractions were three fractions: an upper transparent fraction, an intermediate opaque fraction, and a lower female chromosome fraction. Among the obtained fractions, a mixture of the upper transparent fraction and the intermediate opaque fraction is used as the egg extract. This extract contains no chromosomes, and an egg extract can be obtained from 100 or more unfertilized eggs.
  • the egg extract was prepared just before use. In addition, when there was time restriction, the egg extract prepared in advance was stored at 4 ° C. and used within 2 hours of preparation.
  • an egg extract using egg cells of the same amount as the egg extract used in the first step is newly prepared and added to the sperm-egg extract solution after the second step. 2
  • the mixture was allowed to stand for 30 minutes under a gas phase (third step).
  • the sperm nuclear chromatin could be efficiently induced to the male pronucleus, and the results will be described below.
  • decondensation of sperm nuclear chromatin occurs about 30 minutes after fertilization, histones derived from the fertilized egg are taken into sperm nuclear chromatin in about 45 minutes, and vesicles and nucleoporin sperm about 90 minutes later Binding to nuclear chromatin occurs, vesicle fusion occurs after about 120 minutes, formation of the pronucleus after about 150 minutes, and enlargement of the pronucleus occurs after about 180 minutes.
  • chromatin decondensation occurs about 30 minutes after mixing the sperm nucleus chromatin and the transparent fraction, and between about 30 minutes and 40 minutes later.
  • Histone uptake binding of vesicles and nucleoporins to sperm nuclear chromatin occurs after about 45 minutes, fusion of vesicles after about 90 minutes, formation of pronucleus after about 120 minutes, enlargement of pronucleus after about 150 minutes Occurs.
  • the nuclear process of sperm nuclear chromatin differentiation when treated with an opaque fraction or egg extract decondensation, assembly of nuclear membrane precursor vesicles on the chromatin surface, nuclear membrane
  • the induction rate of fusion of precursor vesicles, formation of pronuclei and enlargement of pronuclei is shown in FIG. 3b.
  • the second step treatment was performed by adding an opaque fraction or egg extract (transparent fraction + opaque fraction) twice as much as the transparent fraction of the first step, the sperm nucleus chromatin
  • the induction rate to the enlarged pronucleus was about 50%.
  • FIG. 5 shows a photomicrograph of the male pronucleus formed as a result of the processing up to the third step.
  • the formed pronucleus is indicated by an arrowhead and an arrow.
  • the right figure of FIG. 5a is an enlarged view of the pronucleus indicated by the arrow in the left figure of FIG. 5a. It was confirmed whether or not a functional nuclear membrane was formed on the pronucleus formed in this way. Normally, low molecular weight substances can migrate into the nucleus through the nuclear pores, but high molecular weight substances cannot pass through the nuclear pores and are not transported into the nucleus.
  • the present technology is expected to be used for evaluating in advance the fertilizing ability of sperm used in, for example, industrial fields such as livestock breeding.

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Abstract

　Cette invention concerne un procédé pour induire la chromatine en pronucléus à l'aide d'un système de reconstitution acellulaire qui utilise un extrait d'ovocytes de mammifères. Plus spécifiquement, l'invention concerne un procédé d'induction de pronucléi à partir de chromatine, le procédé comprenant les étapes (a) à (c) suivantes. (a) Étape 1 Mélange de la chromatine et de la fraction transparente d'un extrait d'œufs préparé à partir d'œufs activés dérivés d'un mammifère, et incubation de ce mélange ; (b) Étape 2 Ajout en outre d'un extrait d'œufs ou de la fraction opaque au mélange de l'étape 1, et incubation de ce mélange ; (c) Étape 3 Ajout en outre d'un extrait d'œufs ou de la fraction opaque au mélange de l'étape 2, incubation de ce mélange, et confirmation que la chromatine dans le mélange s'est différenciée en pronucléi ou non
PCT/JP2015/060344 2014-04-02 2015-04-01 Système de reconstitution acellulaire utilisant un extrait d'ovocytes de mammifères WO2015152330A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004016109A (ja) * 2002-06-17 2004-01-22 Nagoya Industrial Science Research Inst 軟骨細胞様細胞、及びその作製方法
JP2011098900A (ja) * 2009-11-05 2011-05-19 Toagosei Co Ltd 細胞分化誘導ペプチド及びその利用
WO2012173207A1 (fr) * 2011-06-14 2012-12-20 独立行政法人理化学研究所 Procédé d'induction de la différenciation en cellules de la rétine

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004016109A (ja) * 2002-06-17 2004-01-22 Nagoya Industrial Science Research Inst 軟骨細胞様細胞、及びその作製方法
JP2011098900A (ja) * 2009-11-05 2011-05-19 Toagosei Co Ltd 細胞分化誘導ペプチド及びその利用
WO2012173207A1 (fr) * 2011-06-14 2012-12-20 独立行政法人理化学研究所 Procédé d'induction de la différenciation en cellules de la rétine

Non-Patent Citations (4)

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FUKASHI INOUE ET AL.: "Live imaging analysis of sperm chromatin in a cell -free system from mouse oocytes", J. MAMM. OVA. RES., vol. 28, no. 2, 2011, pages S28 *
FUKASHI INOUE ET AL.: "Mouse Ran Chushutsueki o Mochiita Musaibo-kei ni yoru Seishikaku no Keitai Henka no Yudo", THE JAPANESE SOCIETY OF ANIMAL REPRODUCTION KOEN YOSHISHU, vol. 104, 2011, pages 108 *
FUKASHI INOUE ET AL.: "The pronuclear expansion of sperm nuclei induced with the cell -free system from mouse oocytes", J. MAMM. OVA. RES., vol. 29, no. 2, 2012, pages S59 *
YOSHIHIKO HOSOI ET AL.: "Usagi to Ushi ni Okeru Seishi Tobu Kenbi Chunyu ni yoru Taigai Jusei", MEMOIRS OF THE SCHOOL OF BIOLOGY-ORIENTED SCIENCE AND TECHNOLOGY OF KINKI UNIVERSITY, vol. 1, 1997, pages 64 - 71 *

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