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The Birth of Mice from Testicular Spermatozoa Retrieved from Frozen Testicular Sections¹

Laboratory for Genomic Reprogramming, Center for Developmental Biology, RIKEN, Chuo-ku, Kobe 650-0047, Japan

ABSTRACT

Male gamete cryopreservation has been widely used for both human reproduction and animal breeding. We investigated whether testicular spermatozoa retrieved from frozen testicular sections (10 or 25 µm thick) could support the full-term development of normal progeny. For this purpose, frozen testicular sections were prepared from two genetic backgrounds (BDF1 or B6 GFP transgenic mice), and the functional ability of testicular spermatozoa after preservation for 1 day, 1 mo, and 3 mo was assessed by intracytoplasmic sperm injection (ICSI). Testicular spermatozoa were successfully retrieved from frozen testicular sections for the use of ICSI, regardless of the preservation period. The ICSI technique revealed that oocytes (BDF1 or B6 background) injected with testicular spermatozoa prepared from frozen testicular sections developed into normal progeny, even though the sections had been cryopreserved for 3 mo at –30°C. Approximately 15% and 5% of the embryos preserved for 3 mo developed to full term if the testicular spermatozoa were prepared from the 25- and 10-µm sections, respectively. These results clearly indicate that male gametes can be viably preserved in frozen testicular sections. The technique described herein will allow the preservation of male gametes in the form of a "book" or "file" by mounting the sections on a paper-thin sheet. Furthermore, this technique may be of value in the clinical treatment of severe male infertility, since testicular spermatozoa can easily be found through examination of testicular cross sections rather than by attempts to identify them in testicular cell suspension.

fertilization, frozen testicular section, ICSI, spermatid, spermatogenesis, spermatozoa, testicular, testis

¹Supported by grants for Scientific Research in Priority Areas and the Project for the Realization of Regenerative Medicine (research field: technical development of stem cell manipulation) to T.W. by the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

Correspondence: ²Hiroshi Ohta, Laboratory for Genomic Reprogramming, Center for Developmental Biology, RIKEN, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan. FAX: 81 78 306 3095; e-mail: ohta{at}cdb.riken.jp