61 TRANSGENIC HUNTINGTON'S DISEASE MONKEY SPERM HAS A LOWER CRYOTOLERANCE

Abstract

Cryopreservation is an important tool routinely used for preserving sperm for artificial reproductive technologies (ART), as well as genetic preservation of unique animal models. The cryopreservation process is harsh and detrimental to the fragile gametes, and damage to the sperm is not only known, but inevitable. This study presents new data in which sperm from 3 transgenic Huntington's disease (HD) monkeys (rhesus macaques) are compared with 3 wild-type (WT) rhesus sperm donors. Currently, there are no data comparing HD versus WT sperm viability and cryotolerance in humans. The goal of this study was to investigate differences between fresh and frozen semen by quantitative analysis on sperm viability based on (1) motility, (2) membrane integrity, and (3) acrosome integrity. Sperm motility was determined by visual evaluation. Membrane and acrosome integrity were assessed simultaneously by Hoechst 33342, propidium iodide (PI), and fluorescein isothiocyanate-peanut agglutinin (FITC-PNA) triple staining. Sperm viability analysis was divided into 3 groups: (1) fresh HD versus fresh WT, (2) fresh versus cryopreserved-thawed WT, (3) and fresh HD versus cryopreserved-thawed HD sperm. Interestingly, fresh HD sperm had a lower percentage of membrane-damaged cells (38.57 ± 3.15) compared with WT (49.67 ± 3.56; P < 0.03). However, after cryopreservation and subsequent thawing, HD sperm had a significantly higher percentage increase in damaged membranes than WT sperm (27.91 ± 2.93 v. 8.27 ± 8.28; P < 0.001), respectively. No significant difference in acrosome damage between groups was identified in either fresh or cryopreserved sperm populations. Motility significantly declined in both cryopreserved populations [HD: 89.7 to 43.4% (P < 0.001) and WT: 90.0 to 45.6% (P < 0.001)]. There was no significant difference between either freeze-thawed group. These data illustrate that HD sperm have a lower cryotolerance than WT sperm. Our findings suggest that the optimization of the HD sperm cryopreservation method and investigation on biochemical differences (e.g. membrane lipid composition) are necessary to improve post-thaw survival. This in turn is important for the establishment of a sperm cryobank and future derivation of a unique animal model such as HD monkey. Our study also suggests that HD monkey could be a useful model for optimizing cryopreservation method for HD patients.