TESTICULAR SPERMATOGENESIS, MORPHOGENESIS, AND GENE EXPRESSION
7:30 AM-10:00 AM
(479) CRYOPRESERVATION OF PORCINE TESTICULAR TISSUE.
Snedaker, Amy1, Honaramooz, Ali1, Schlatt, Stefan2, Dobrinski, Ina1, 1 Center for Animal Transgenesis and Germ Cell Research, Kennett Square, PA2 Institute of Reproductive Medicine, Muenster, Germany
ABSTRACT- Preservation of the male germ line may be accomplished by freezing sperm or, in situations where mature sperm cannot be obtained, by cryopreservation of testis cells or tissue. Cryopreservation of testis tissue has the advantage of maintaining the structural integrity and providing the compatible microenvironment needed for completion of spermatogenesis after xenotransplantation. A protocol for the cryopreservation of porcine testicular tissue has not been established. The objective of this study was to compare the viability and developmental potential of porcine testicular tissue after preservation by different methods of cryopreservation or short-term refrigeration. Small fragments of testis tissue from 1-wk old piglets and 3-mo old boars were prepared. Four preservation methods included: 1) Automated slow freezing protocol involving immersion in Leibovitz medium containing 2% bovine serum albumin and 10% dimethyl sulfoxide (DMSO), cooling at -2°C/min from 20°C to 4°C; -0.3°C/min from 4°C to -30°C; and -50°C/min from -30°C to -130°C, and storing in a liquid N2 freezer; 2) Conventional slow freezing protocol established for isolated germ cell suspensions using fetal bovine serum (FBS), Dulbecco Modified Eagle Medium, and DMSO at a 1:3:1 ratio, and a Nalgene alcohol bath container to provide a rate of -1°C/min to -70°C, before storing in a liquid N2 freezer; 3) Vitrification involving processing through a solution containing DPBS with 10% FBS, 10% glycerol and 20% ethylene glycol, then DPBS with 10% FBS, 25% glycerol and 25% ethylene glycol, 15 min each, followed by plunging into LN2; 4) Refrigeration at 4°C for 2 days. Trypan blue exclusion was used to assess viability of single cell preparations after enzymatic digestion of the preserved testis fragments. Viability of cells from the vitrification protocol was less than 33%, while that of the other protocols was more than 60%. Four weeks after transplantation into immunodeficient mice, graft survival with subsequent differentiation was observed for all protocols except vitrification. In all groups with surviving grafts, graft size increased more dramatically with newborn donor tissue. These results indicate that porcine testis tissue can be successfully stored by refrigeration for 2 days or cryopreserved using slow-freezing protocols prior to transplantation.
KEY WORDS: cryopreservation, testis, pig