25 Cryopreservation of horse testicular tissue as a model for rhinoceros

Abstract

Cryopreservation of testicular tissue (TT) allows retention of valuable genetic material that can be used for conservation of endangered species, such as the northern white rhinoceros (NWR; Ceratotherium simum cottoni). Previously, we found that cryopreservation of NWR TT with a slow controlled cooling rate (CR) method induced morphological alterations in the seminiferous tubules (ST). However, the relative influence of CR, type of medium, and condition of TT from the aged NWR male on TT integrity was not clear. Due to the limited availability of rhinoceros TT, we used the horse as a model for optimization of TT cryopreservation. We evaluated the effect of (1) cryoprotectant solution [PBS (PBS +1.5 M dimethyl sulfoxide) v. DMEM (DMEM/F12 + 10.0% fetal bovine serum + 0.05 M sucrose + 1.5 M dimethyl sulfoxide)] and (2) CR [CR1 (−2.0°C min⁻¹ from 0°C to −4.0°C, −15°C min⁻¹ to −12°C, and −0.3°C min⁻¹ to −40°C in a programmable freezer) v. CR2 (same as CR1 but cooled to −8°C and held for 5 min before cooling to −40°C) v. CR3 (−1.0°C min⁻¹ from 0°C to −80°C in a CoolCell® freezing device; Corning, Corning, NY, USA)] on the structural integrity of ST from a 2-year-old horse (n = 20 ST), cell viability, and expression of spermatogonial stem cells (SSC; GFRα1, and GRP125) and pluripotent markers (SSEA-4, SSEA-1, and OCT-4) in spermatogonial cells isolated from TT frozen with the above treatments (n = 3). We found a positive interaction between CR and cryoprotectant solution on structural integrity of fixed and stained TT after freezing in PBS and CR2 that resulted in lower detachment of epithelium cells from the basement membrane (score ± standard error of the mean; 0.50 ± 0.1) than that of TT frozen in PBS and CR1 and CR3 (1.00 ± 0.1 and 1.80 ± 0.1, respectively; P < 0.001) or in DMEM and CR1 (1.25 ± 0.1), CR2 (1.35 ± 0.1), and CR3 (1.40 ± 0.1; P < 0.01) and in lower incidence of basement membrane damage (0.75 ± 0.1) than that of TT frozen in PBS and CR1 (1.17 ± 0.07) and CR3 (1.16 ± 0.07) or in DMEM and CR1 (1.10 ± 0.1), CR2 (1.15 ± 0.1), or CR3 (1.45 ± 0.1; P < 0.01). A lower rate of pyknosis was observed in TT frozen with PBS (1.15 ± 0.06) than in TT frozen in DMEM (1.43 ± 0.06; P < 0.001). Overall, integrity of ST was improved when TT was frozen in PBS at CR2 having similar percentages of ST with intact epithelium (60%) and basement membrane (35%) as that of refrigerated TT (45 and 50%, respectively) but different from that of TT frozen with PBS at CR1 (10 and 15%, respectively; P < 0.05). Flow cytometry analysis of spermatogonial cells revealed that the percentages of live cells from TT frozen in PBS (CR1: 61.5 ± 7.4%; CR2: 59.7 ± 4.8%; CR3: 51.5 ± 4.1%) or DMEM (CR1: 66.2 ± 6.0%; CR2: 59.8 ± 6.0%; CR3: 58.9 ± 6.9%), and expression of SSC and pluripotent markers was similar among all freezing treatments. However, the percentages of live cells from frozen-thawed TT were lower than those of cells isolated from refrigerated TT (80.6 ± 2.2%; P < 0.001). Overall, our results showed that (1) structural integrity of horse ST was better maintained when TT was frozen in PBS at CR2 and (2) SSC can be isolated from frozen-thawed TT with a similar relative frequency to that of refrigerated TT.