We reported here that the spermatozoa and spermatids collected from frozen epididymis and testis or from frozen bodies of mice were able to produce normal offspring after injection into oocytes. Obviously, those frozen spermatozoa and spermatids were all "dead" in the conventional sense, as confirmed by propidium iodide staining. Their plasma membranes were severely damaged. Motionless spermatozoa had no chance whatsoever of fertilizing oocytes in vivo or in vitro. Nevertheless, some of these spermatozoa and spermatids, if not all, were "alive" or "genomically intact," because they were able to produce apparently normal offspring.
Freezing protocols used here are very simple. Simple freezing of animal bodies, using ordinary freezers, is perhaps the simplest procedure we can think of, and it can be done in any laboratory. Isolation and freezing of testes and epididymides are also simple and can be done in most laboratories or even in the field. Our protocols require a set of micromanipulators to generate offspring, but microsurgical injection of spermatozoa (or spermatogenic cells) into oocytes is no longer a special procedure. In fact, thousands of infertility clinics throughout the world routinely perform microsurgical sperm injection to overcome various types of male infertility (14, 15). Normal births after microinsemination have been reported in 14 mammalian species, as of today (11). Laboratories that do not have microsurgical facilities or experience may keep the frozen testis/epididymis or entire bodies in an ordinary freezer (without an automatic defrosting cycle) until they are ready to send the specimens to other laboratories, where microinsemination is routinely performed. This would be particularly pertinent when precious male animals died unexpectedly.
Of all mouse strains, C57BL/6 (B6) would receive the most benefit from this freezing procedure. B6 mice have been most extensively used as a standard strain of mouse for genetic studies. Many genetically modified mice have a B6 background. Difficulty in cryopreserving their spermatozoa has been a major obstacle in mouse genetics (2, 16). Here we demonstrated that B6 mice can be produced by using spermatozoa retrieved from testes frozen for 5–7 months or air-transported with dry ice. The freezing protocols reported here are simple and cost-effective. They would enhance exchange of mouse genetic resources among many laboratories around the world.
It was rather unexpected that spermatozoa in the testes would withstand freezing better than those in the epididymis (Table 3), because nuclei of epididymal spermatozoa are known to be much more stable, both physically and chemically, than testicular spermatozoa because of extensive crosslinkings of nuclear protamines by disulfide bonds (17, 18). Our two-factorial statistical analysis, consisting of three germ cell types and four freezing protocols (Table 1), revealed that epididymal spermatozoa were most sensitive to freezing of all germ cells examined. The reason for this is not clear, but luminal fluid in the seminiferous tubule and/or some materials from Sertoli or spermatogenic cells might have contributed to alleviating sperm damage by freezing. Although round spermatids recovered from defrosted testes can be used to produce live offspring (Tables, 1, 2, and 4), selective identification of these cells from other cells could be rather difficult for inexperienced researchers. Elongated spermatids or spermatozoa are obviously much easier to identify (Fig. 2).
One thing to be stressed here is that the type of medium used for suspension of defrosted spermatozoa and spermatids makes a difference in the outcome of the experiments: the birth of normal offspring. Potassium-rich Ca2+- and Mg2+-free NIM medium (13, 19) always gave better results (Tables 2 and 3). Ca2+-containing ordinary cell culture media like PBS may activate endogenous nucleases (20), which attack the DNAs of these spermatozoa and spermatids with broken plasma membranes. Cauda epididymal spermatozoa may have a higher nuclease activity than testicular spermatozoa and spermatids, and this may make the former more sensitive to freezing and thawing than the latter.
The present study has shown that spermatozoa and spermatids can retain their fertilizing ability in frozen reproductive organs or whole bodies for longer than we anticipated. We found that the spermatozoa retrieved from the testes of mice frozen at –20°C for 15 years were able to produce normal offspring by microinsemination. It would be interesting to know the optimal temperature to use for whole-body freezing and how long male germ cells can retain their fertilizing ability. Accelerated degradation kinetics that have been applied to estimate the maximum storage period of freeze-dried mouse spermatozoa (21) may be applicable to answer this question. If spermatozoa of extinct mammalian species (e.g., woolly mammoth) can be retrieved from animal bodies that were kept frozen for millions of years in permanent frost, live animals might be restored by injecting them into oocytes from females of closely related species.