358 MICROINSEMINATION USING MALE GERM CELLS FROM EPIDIDYMIDES AND TESTES STORED IN FREEZERS WITHOUT CRYOPROTECTANT

Abstract

Cryopreservation of male germ cells is a strategy for the conservation of species and strains valuable to biomedical researchers. However, to minimize damage that may occur during freezing and thawing, complex cryopreservation protocols that have been optimized for the stage and species of the male germ cell are usually employed. This study was undertaken to see whether mouse male germ cells could be safely cryopreserved for later use by freezing the whole epididymides and testes without cryoprotectant. Furthermore, we examined whether frozen male germ cells maintained their fertilization ability after international transportation on dry ice. Epididymides and testes were collected from sexually mature male ICR and C57BL/6Cr mice and placed in polypropylene cryotubes. The cryotubes were frozen at -80°C with or without a freezing container, or were plunged directly into liquid nitrogen (LN₂). They were stored at -80°C or in LN₂ from between one week and one year. Epididymides and testes were thawed by placing the cryotubes in a water bath at room temperature. B6D2F1 and C57BL/6Cr oocytes were microinseminated with either epididymal and testicular spermatozoa or round spermatids. After embryo transfer into pseudopregnant females, normal pups were obtained irrespective of the method of cryopreservation and cell type used. However, their birth rates (2-33%) were lower than those of our conventional microinsemination using fresh sperm or spermatids (20-60%). For transportation experiments, testes were collected from C57BL/6J mice and placed in a cryotube. The cryotubes were frozen at -80°C in a freezing container. On the day of transportation, the cryotubes were placed in a polystyrene foam case filled with dry ice and were transported from Harwell (UK) to Tsukuba (Japan) by air and land. After three days, the samples were delivered to the recipient facility and were stored at -80°C until use (about 1 month). After thawing and collection of spermatogenic cells, C57BL/6J oocytes were microinseminated with either testicular spermatozoa or elongated spermatids. After embryo transfer, 24 (34% per transfer) and 8 (16%) offspring, respectively, were obtained from the two groups. These results indicate that mouse male germ cells retain their nuclear integrity even after freezing epididymides or testes in freezers without cryoprotectant. Since this cryopreservation technique is very simple and allows storage at -80°C for at least several months, it may enable transportation of mouse male germ cells internationally on dry ice, even when the senders are not specialized in cryopreservation.