In fertilization, when a sperm approaching an ovum comes into contact with the zona pellucida of the ovum, an acrosome reaction occurs and acsorome at the anterior part of the head changes to release lytic enzymes such as hyaluronidase, acrosin and the like, and owing to the actions of these enzymes, the sperm passes through zona pellucida and reaches the ovum cell present inside, attaining fertilization. Dysplastic sperms and cryopreserved sperms, however, have lowered fertilizing capacity, and it is difficult to produce a fertilized ovum with these sperms in usual in vitro fertilization in which an ovum and sperms are only mixed. Therefore, there are various developed technologies for enhancing the fertilization rate in in vitro fertilization of mammals.
Now, analysis of gene functions and associated researches and developments are actively conducted worldwide as national projects, and genetically-modified (Tg (Transgenic) and KO (Knock Out)) mice play an important role in them. Since KO mice are produced in sequence, the number of them is believed hereafter to exceed 1000000 kinds, and for keeping their lineages, enormous amount of mice should be bred, and maintenance management of these mice is an extremely serious problem in laboratory animal facilities around the world. So, preservation of these mice by frozen sperms is attracting attention, however, especially sperms of C57BL/6 mice generally used as laboratory animals are often damaged by a freezing treatment and a thawing treatment to lower vital energy and deficient in force for passing through zona pellucida, thus, the fertilization rate thereof is low.
Recently, an improved method for freezing sperms of C57BL/6 mice has been reported (non-patent document 1), however, it is difficult to obtain stable high fertilization rate since this method includes complicated operations, and generally, a fertilization rate of only 10 to 20% is obtained under present circumstances.
Further, a cryopreserved ovum is used in in vitro fertilization when use of a fresh ovum is difficult, however, since zona pellucida hardens by freezing and thawing, there is a problem of lowering of fertilization rate with a freeze-thawed ovum even if fresh sperms are used.
Under such conditions, assisted reproduction technologies as described below are presently used for efficient production of a fertilized ovum in in vitro fertilization using sperms having lowered fertilizing capacity or in in vitro fertilization using a cryopreserved ovum.
For sperms having relatively kept motility, zona pellucida partial dissection methods (PZD: Partial Zona-Pellucida Dissection (non-patent document 2); ZIP: Partial Zona-Pellucida Incision by Piezo-micromanipulator (non-patent document 3)) and a laser zona pellucida drilling method (patent document 1) are used. In the zona pellucida partial dissection method, the zona pellucida of an ovum is cut using a point of a syringe needle (PZD) and a point of a glass capillary tube connected to a Piezo-micromanipulator, and in the laser zona pellucida drilling method, the zona pellucida of an ovum is drilled by laser. Sperms become able to break in an ovum through slits or pores made on zona pellucida, to enhance fertilization rate, by these methods.
In contrast, for sperms showing no motility at all after freezing and thawing, an intracytoplasmic sperm injection method (ICSI: Intra Cytoplasmic Sperm Injection) in which one sperm is mechanically injected directly into ooplasm (non-patent document 4) is used in general.
However, these methods need special technologies and apparatuses and thus cause a problem of low versatility.
Zona pellucida has also a function of protecting a fertilized ovum (fertilized embryo). Zona pellucida is a membrane present around an ovum, and a fertilized ovum is enclosed and protected by zona pellucida and grows under this condition until predetermined time, and then, break away from zona pellucida and nidate.
However, implantation into uterus may be impaired when an embryo in the early stages of development (blastocyststage embryo) cannot hatch zona pellucida in the development stage. Such an impairment tends to occur when an ovum having dysplastic zona pellucida is used, when in vitro fertilization is conducted using a cryopreserved ovum or when a fertilized ovum (embryo) is cryopreserved, then, thawed and implanted into uterus, in addition to the case of hardening or thickening of the zona pellucida of an ovum obtained in maturing a follicle using an ovulation inducing drug.
For a fertilized ovum obtained by in vitro fertilization to easily nidate, an assist hatching method (zona drilling) is used before implantation into uterus. The assist hatching method includes a zona pellucida partial dissection method for mechanically cutting zona pellucida using a point of a syringe needle or a glass capillary tube (non-patent documents 2 and 3), a method for drilling zona pellucida by laser (patent document 1) and a method for lysing zona pellucida with an enzyme (non-patent document 5)
However, the method of using a point of a syringe needle or a glass capillary tube and the method for drilling zona pellucida by laser need special apparatuses and technologies and have a problem of low versatility. The method with an enzyme has a problem of lowering of a developmental ability due to damage on an embryo.
Genetically modified animals, for example, genetically-modified mice (knockout mice) are generally produced by the following procedures. First, targeting vectors (recombinant DNA) are prepared, then, the targeting vectors are introduced into ES cells by an electroporation method and the like. ES cell strains which have undergone homologous gene recombination are selected. Next, a chimera is produced by a method in which ES cells are injected by using a micro-manipulator into a cavity formed in a fertilized ovum at the blastocyst stage to produce a chimera embryo, and this chimera embryo is transplanted into the uterus of a pseudo-pregnant mouse (injection chimera production method) and a method in which an embryo in the early stages of development (for example, 8-cell stage embryo) from which zona pellucida has been eliminated is produced, ES cells are added to this and an aggregated lump is made, which is incubated until the blastocyst stage and transplanted into the uterus of a pseudo-pregnant mouse (aggregation chimera method). Then, offsprings (chimera mice) are obtained. Next, the produced chimera mouse and a wild type mouse are mated, and it is confirmed whether the germ cells are formed by cells derived from recombinant ES cells. Then, mice in which formation of the germ cells by cells derived from recombinant ES cells has been confirmed are mutually mated, and knockout mice are selected from the resultant offsprings.
The micro-manipulator method used in the above-described method needs special apparatuses and technologies. By contrast, in the aggregation chimera method, elimination of zona pellucida of an embryo in the early stages of development is necessary, and Acidic Tyrode's solution (pH 2) is used for elimination of zona pellucida (patent document 2). When Acidic Tyrode's solution is used, there is a problem of high cytotoxicity of the Acidic Tyrode's solution itself, and there is a problem that it should be used under protein-free condition during the operation and an embryo in the early stages of development adheres to a dish or a glass capillary to lower operability extremely.
In addition to the above-described assisted reproduction technologies, those related to culture media and culturing methods are suggested such as a method for pre-incubating sperms in a culture medium containing a cyclodextrin derivative and an amino acid and/or a glycolytic intermediate substance for raising fertilization rate (patent document 3), a method in which an unfertilized ovum is added to sperms pre-incubated in a reduced glutathione-containing culture medium and fertilization is performed in the presence of reduced glutathione, to prevent lowering of fertilization rate due to hydrogen peroxide generated in using freeze-thawed sperms (non-patent document 6), and a continuous culturing system and a process for enhancing pregnancy efficiency in human in vitro fertilization (patent document 4), and the like.