A decellularized oocyte-derived scaffold provides a "sperm safe" to preserve mammalian spermatozoa.

BACKGROUND: Although only one spermatozoon is needed to create a zygote, a significant challenge is the storage and recovery of germ cells when sperm counts are extremely low. OBJECTIVES: We engineered an oocyte-derived biomaterial-the zona pellucida (ZP)-as a "sperm safe" for storing spermatozoon. The ZP is a glycoprotein matrix that surrounds the mammalian oocyte. MATERIALS AND METHODS: We made a hole in the ZPs using a Piezo drill and mechanically separated them from the oocyte cytoplasm. A subset of ZPs were further purified through decellularization. Using a modified ICSI approach, we injected sperm heads into purified ZPs and tested the efficacy of cryopreservation and recovery of spermatozoon as well as function. RESULTS: Between 1-6 sperm heads were injected into purified ZPs (average 2.7 ± 1.7 sperm heads/ZP), which were then cryopreserved. Upon thawing, an average of 2.5 ± 1.4 sperm heads/ZP were observed, and in 11 of 12 thawed "sperm safes," we recovered all spermatozoa. Decellularized "sperm safes" maintained their three-dimensional structure and had a denser matrix relative to untreated controls as assessed by scanning and transmitted electron microscopy. The efficacy of "sperm safe" derived spermatozoon was evaluated by ICSI. Spermatozoon stored in either untreated or decellularized "sperm safes" elicited egg activation-associated calcium transients and zinc sparks when injected into eggs. Of the resulting zygotes, >80% of them formed pronuclei irrespective of the sperm source. 26.8 ± 4.6% and 18.1 ± 7.0% of the pre-implantation embryos generated from spermatozoon recovered from untreated or decellularized "sperm safes" developed to the blastocyst stage, respectively. Although this development was lower than that using fresh spermatozoon (59.3 ± 19.3%) or conventionally frozen-thawed spermatozoon (28.4 ± 1.7%), these differences were not significant. DISCUSSION AND CONCLUSION: Purified ZPs represent a natural biomaterial for the efficient preservation and recovery of small sperm numbers.