such as "Introduction", "Conclusion"..etc
Narumi Ogonuki*, Keiji Mochida*, Hiromi Miki*, Kimiko Inoue*, Martin Fray, Takamasa Iwaki, Kazuo Moriwaki*, Yuichi Obata*, Kazuto Morozumi, Ryuzo Yanagimachi,¶, and Atsuo Ogura*,¶
*Institute of Physical and Chemical Research (RIKEN) Bioresource Center, Tsukuba, Ibaraki 305-0074, Japan; Medical Research Council Mammalian Genetics Unit, Oxfordshire, OX11 0RD, United Kingdom; Jikei University School of Medicine, Tokyo 105-8461, Japan; and Institute for Biogenesis Research, University of Hawaii School of Medicine, Honolulu, HI 96822
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 C57BL/6). Epididymides and testes frozen and transported internationally to another laboratory by air could produce pups of inbred C57BL/6 mice. Testicular spermatozoa retrieved from the bodies of male mice (BALB/c nude and C3H/He 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
Sperm 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 C57BL/6 (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 frozen-thawed 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 frozen reproductive organs or frozen bodies of mice, can produce offspring by microinsemination.
Source: PNAS, August 29, 2006, vol. 103, no. 35, 13098-13103.
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