Development of Normal Mice from Oocytes Injected with Secondary Spermatocyte Nuclei1

Kimura, Yasuyuki; Yanagimachi, Ryuzo

doi:10.1095/biolreprod53.4.855

Cited by 207 publications

(111 citation statements)

References 32 publications

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“…Recipient blastocysts for chimera production were obtained by in vitro fertilization of (C57BL͞6 ϫ DBA͞2)F 1 (BDF1) oocytes with B6 sperm. Cells were microinjected into the blastocoels by using a Piezo-driven micromanipulator (43). Microinsemination was carried out by using BDF1 oocytes, as described in ref.…”

Section: Methodsmentioning

confidence: 99%

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Production of knockout mice by random or targeted mutagenesis in spermatogonial stem cells

Kanatsu-Shinohara

Ikawa

Takehashi

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

145

108

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Stem cells represent a unique population of cells with self-renewal capacity. Although they are important therapeutic targets, the genetic manipulation of tissue-specific stem cells has been limited, which complicates the study and practical application of these cells. Here, we demonstrate successful gene trapping and homologous recombination in spermatogonial stem cells. Cultured spermatogonial stem cells were transfected with gene trap or gene targeting vectors. Mutagenized stem cells were expanded clonally by drug selection. These cells underwent spermatogenesis and produced heterozygous offspring after transplantation into the seminiferous tubules of infertile mouse testes. Heterozygous mutant mice were intercrossed to produce homozygous gene knockouts. Using this strategy, the efficiency of homologous recombination for the occludin gene locus was 1.7% using a nonisogenic DNA construct. These results demonstrate the feasibility of altering genes in tissue-specific stem cells in a manner similar to embryonic stem cells and have important implications for gene therapy and animal transgenesis.spermatogenesis ͉ germ cell ͉ testis ͉ transplantation S tem cells represent a unique cell population with self-renewal potential (1). Although stem cells are low in number, these cells proliferate extensively to sustain the various self-renewing tissues, such as bone marrow and intestine. Although these tissue-specific stem cells normally divide very slowly, stem cells are the last cell type to be destroyed after cytotoxic damage, and they regenerate the entire tissue in a relatively short time. In addition, stem cells often have migratory activities, and they can be transplanted between animals; transplanted stem cells migrate to a specific niche and regenerate the self-renewing tissue. Because of their unique properties, stem cells have become the attractive target of cell and gene therapies.Among the many types of tissue-specific stem cells, spermatogonial stem cells are unique in that they have germ-line potential (2, 3). Genetic modification of spermatogonial stem cells creates permanent changes in the germ line, which are transmitted to the offspring by means of fertilization. In contrast to female germ-line cells, which cease to divide after birth, male germ-line cells proliferate continuously and produce sperm throughout the life of the animal. If these stem cells could be cultured and manipulated in a manner similar to embryonic stem (ES) cells (4, 5), they could be used to create knockout animals. As a first step toward this goal, a germ cell transplantation technique was developed in 1994 (6, 7), in which dissociated donor testis cells colonized the seminiferous tubules of infertile recipient testis and produced donor-derived spermatogenesis and offspring. Although this technique was an opportunity to produce offspring from manipulated spermatogonial stem cells, it has been difficult to produce transgenic animals using spermatogonial stem cells, because their number is very low in the testis, and the lack of me...

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Section: Methodsmentioning

confidence: 99%

“…Microinsemination was carried out by using BDF1 oocytes, as described in ref. 43. The embryos were transferred into the oviducts of ICR pseudopregnant females on the day after culture.…”

Section: Methodsmentioning

confidence: 99%

Production of knockout mice by random or targeted mutagenesis in spermatogonial stem cells

Kanatsu-Shinohara

Ikawa

Takehashi

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

145

108

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“…Intracytoplasmic injection of spermatozoa and spermatids was performed by using a micropipette attached to a Piezo-electric actuator (Prime Tech, Ibaraki, Japan), as described (24,25). The cover of a plastic dish (50 ϫ 3 mm; Falcon no.…”

Section: International Transport Of Frozen Epididymides and Testesmentioning

confidence: 99%

Spermatozoa and spermatids retrieved from frozen reproductive organs or frozen whole bodies of male mice can produce normal offspring

Ogonuki

Mochida

Miki

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Cryopreservation of male germ cells is a strategy to conserve animal species and strains of animals valuable to biomedical research. We tested whether mouse male germ cells could be cryopreserved without cryoprotection by simply freezing epididymides, testes, or whole bodies. The reproductive organs were isolated from killed mice and frozen for 1 week to 1 year at ؊80°C before spermatozoa and spermatids were collected and injected into mature oocytes. Normal pups were born irrespective of strains tested (ICR and C57BL͞6). Epididymides and testes frozen and transported internationally to another laboratory by air could produce pups of inbred C57BL͞6 mice. Testicular spermatozoa retrieved from the bodies of male mice (BALB͞c nude and C3H͞He strains) that had been kept frozen (؊20°C) for 15 years could also produce normal offspring by microinsemination. Thus, freezing of either male reproductive organs or whole bodies is the simplest way to preserve male germ cells. Restoration of extinct species could be possible if male individuals are found in permafrost.cryopreservation ͉ gametes ͉ intracytoplasmic sperm injection ͉ microinsemination ͉ mouse S perm cryopreservation has been widely used for both human reproduction and animal breeding. Because much of basic research of mammalian genetics and early development has been done by using laboratory mice, and the number of genetically engineered mice (transgenesis, gene targeting, and mutagenesis) is increasing exponentially, development of simple, cost-saving, and space-effective means of mouse sperm preservation is much needed (1). Mouse sperm cryopreservation using raffinose and skim milk as cryoprotectants has been successful, but defrosted spermatozoa of some strains of mice do not fertilize eggs well (2). This is especially true for C57BL͞6 (B6) mice, most frequently used for generation of genetically engineered mice. This problem has been overcome by partial zona dissection before in vitro fertilization (3) or intracytoplasmic sperm injection of frozenthawed or freeze-dried spermatozoa (4-7).Immature male germ cells such as spermatids and spermatocytes are currently used to produce offspring (8-11) when spermatozoa cannot be obtained due to spermatogenic arrest because of genetic mutations or as the result of in vitro manipulation of germ cells (7, 10, 11). Therefore, cryopreservation of these immature sperm cells is necessary, but they suffer more cryodamage than mature epididymal spermatozoa. A cryopreservation protocol we developed for mouse spermatogenic cells in the past is rather complicated, and therefore the development of simpler techniques is required (12).Thus far, the simplest method of cryopreserving mouse spermatozoa is to freeze spermatozoa in simple salt solutions without any cryoprotectants (6). Although defrosted spermatozoa are not alive in the conventional sense, they apparently maintain their genetic integrity, because they are able to produce live offspring by microinsemination. Here we report that spermatozoa or spermatids, retrieved from f...

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“…If the oocyte is activated, the disappearance of MPF and CSF activity will lead the injected cell nucleus to undergo condensation and to complete meiosis and form a haploid chromosome set. Thus, the premature chromosome condensation of injected cells occurring in the absence of oocyte activation is essential for achieving fertilization with secondary spermataocytes (Kimura and Yanagimachi 1995).…”

Section: Use Of Secondary Spermatocytesmentioning

confidence: 99%

Advances in Fertility Options of Azoospermic Men

Wu¹,

Gelety²,

Shi³

2012

Advances in Embryo Transfer

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Development of Normal Mice from Oocytes Injected with Secondary Spermatocyte Nuclei1

Cited by 207 publications

References 32 publications

Production of knockout mice by random or targeted mutagenesis in spermatogonial stem cells

Production of knockout mice by random or targeted mutagenesis in spermatogonial stem cells

Spermatozoa and spermatids retrieved from frozen reproductive organs or frozen whole bodies of male mice can produce normal offspring

Advances in Fertility Options of Azoospermic Men

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