Effects of cryopreservation on mitochondrial function and heterogeneity, lipid raft stability and phosphatidylserine translocation in koala (Phascolarctos cinereus) spermatozoa

Abstract

Koala sperm mitochondria were examined by cryomicroscopy using the fluorescent probe JC-1, which distinguishes high (red) and low (green) mitochondrial membrane potential (MMP). At normal body temperature, approximately 70% of live and untreated spermatozoa exhibited high MMP whereas <3% of live untreated spermatozoa exhibited low potential. A third class, in which single midpieces contained mixed mitochondrial populations, was also detected. Heterogeneity was noted in the level of MMP between individual koalas, individual spermatozoa and even between mitochondrial gyres within single midpieces. MMP of the live sperm population was not significantly affected by glycerol but was suppressed by freezing and thawing treatments. After thawing, MMP declined significantly during rewarming, especially as the temperature increased from 5 to 35 degrees C. The distribution of the ganglioside GM1 was examined using fluorescent-labelled cholera toxin B. In fresh, untreated koala spermatozoa GM1 was detected on the head and midpiece, but not on the principal piece. No significant redistribution of GM1 was observed after chilling and cryotreatment. Phosphatidylserine translocation across the plasma membrane was examined using fluorescent-labelled annexin V. Few fresh spermatozoa exhibited phosphatidylserine translocation (approximately 1%); this was not increased by chilling or cryopreservation, thus implying that cryotreatment had little effect on plasma membrane lipid asymmetry.

Osmotic stress and cryoinjury of koala sperm: an integrative study of the plasma membrane, chromatin stability and mitochondrial function

“…This observation suggests that the physiology that drives mitochondrial function is capable of tolerating hypertonic environments better than they can cope with hypotonic changes. Interestingly, the percentage of koala spermatozoa with high MMP following cryopreservation (this study; Zee et al 2007Zee et al , 2009a) was similar to that of spermatozoa exposed to hypotonic conditions and, therefore, suggests that the thawing component of cryopreservation may be most detrimental to koala sperm mitochondria.…”

“…Mitochondrial membrane potential (MMP) has been shown to be a sensitive indicator of the bioenergetic functions within the mitochondrion (Duchen 2004), and while Zee et al (2007Zee et al ( , 2009aZee et al ( , 2009b have previously used the JC1 probe (5,5 0 ,6,6 0 -tetrachloro-1,1 0 ,3,3 0 -tetraethylbenzimidazolyl carbocyanine iodide) to investigate the effect of cryoinjury on the koala sperm midpiece, the relative detrimental effects of osmotic stress vs structural damage associated with intracellular ice crystal damage remain unresolved. While the response of spermatozoa to the damaging effects of osmotic stress during cryopreservation can be demonstrated artificially by exposure of spermatozoa to a range of osmotically defined media (Curry & Watson 1994, Meyers 2005, Willoughby et al 1996, to our knowledge, this experimental model has never been used before to examine the effect of osmosis on sperm DNA, and certainly not for marsupial sperm chromatin.…”

Gamete membrane microdomains and their associated molecules in fertilization signaling

“…The presence of functional MDs in sperm and/or their possible involvement in capacitation have been demonstrated in several animals including mammals (boar, goat, human, koala, mouse, ram, rat), a tunicate (ascidian), and sea invertebrates (sea urchin, starfish) (Ohta et al, 2000; Trevino et al, 2001; Cross, 2004; Shadan et al, 2004; Shoeb et al, 2004; Thaler et al, 2006; Companyo et al, 2007; Zee et al, 2007; Asano et al, 2009; Botto et al, 2010; Konno et al, 2010; Naruse et al, 2010; Nixon et al, 2010). While the presence of a canonical caveolar structure (e.g., rather static invagination) in mouse sperm membranes is still debated, caveolin 1‐containing membrane MDs have been found in guinea pig and mouse sperm (Travis et al, 2001).…”

New insights into the reproductive physiology and management of the female koala (Phascolarctos cinereus): factors affecting the control of the oestrous cycle

“…Despite significant advances in koala sperm cryopreservation (Johnston et al, 2006, Zee et al, 2007, Johnston et al, 2012, Johnston et al, 2013c, the quality of cryopreserved koala semen after thawing is still inadequate for successful AI. Although this precludes genome resource banking for the moment, chilled semen storage offers significant 1-5 logistical flexibility for the application of AI in the koala, particularly as it relates to the national and global movement of genetic material.…”

“…Previous studies (Zee et al, 2007, Johnston et al, 2012, Johnston et al, 2013) have failed to develop an effective method for the cryopreservation of koala spermatozoa to assist with genetic exchange, so AI protocols incorporating oestrous synchronization with the use of chilled semen are required in the short-term. The primary objective of this study was to investigate the potential application of the synthetic GnRH antagonist, azaline B, to synchronize oestrus in the 5-4 koala.…”