Comparison of Membrane Characteristics between Freshly Ejaculated and Cryopreserved Sperm in the Chicken

Abstract

Cryopreserved sperm undergoes serious damage which affects its fertilizing ability. Despite progress in understanding the nature of functional deterioration in mammalian sperm, little is known about the mechanism involved in the induction of functional damage in avian sperm. Cellular membranes are considered the primary site of cryodamage to sperm. Membrane rafts are specific membrane regions enriched in sterols, ganglioside G M1 , and functional proteins and they play important roles in the regulation of diverse functions exerted in mammalian sperm during fertilization. Several reports investigating cryopreservation-induced membrane changes in mammalian sperm have suggested that cryopreservation induces a compositional alteration of membrane rafts via a loss of membrane sterols, leading to impaired fertilizing ability. Recently, we demonstrated that membrane rafts are present in chicken sperm. Therefore, we investigated a possible mechanism for the induction of functional damage in cryopreserved chicken sperm, with particular attention to cryopreservation-induced compositional changes in membrane rafts. Sterol quantification showed that loss of sterols from sperm membranes occurred following cryopreservation. Biochemical analyses of detergent-insoluble membranes showed that the lipid and protein compositions of membrane rafts were altered dramatically by cryopreservation. To determine the physiological role of these changes, we examined external translocation of phosphatidylserine (PS), representing an early apoptotic change, and found that cryopreservation induced apoptotic changes in chicken sperm. Furthermore, methyl-β-cyclodextrin-induced loss of sterols from the plasma membranes stimulated PS translocation that was not accompanied with caspase-3 activation, which plays an important role downstream of the apoptotic cascade. Based on the results obtained in this study, we discuss a new mechanism for reduction of the fertilizing ability in avian sperm after cryopreservation.

Characterization of the functions and proteomes associated with membrane rafts in chicken sperm

“…Similarly, the localization of sterols using Filipin showed a sharp decrease in sterol content in the membranes of cryopreserved sperm. In accordance with our previous findings that cryopreservation-induced sterol loss causes an early apoptotic events [ 21 ], resulting in a decline of fertilizing ability in chicken sperm [ 29 ], the results of present study suggest that a compositional rearrangement of membrane rafts specifically occurs in response to cryopreservation-induced sterol loss, leading to the functional damage in chicken sperm via an induction of an early apoptosis. However, G M1 quantification showed no difference in the low density DRM, although there was a tendency for decreasing the amount in the fraction with a lower buoyant density, such as in fractions 4 and 5.…”

“…Although membrane rafts have been isolated from mammalian sperm, their biochemical and functional properties in avian sperm have not been characterized. Previously, we found in chicken sperm that cryopreservation induced the loss of cholesterol from the membranes, leading to an early apoptotic event [ 21 , 29 ]. Considering other studies in culture cells which have demonstrated that cholesterol loss from membrane rafts induced early cellular apoptosis [ 30 – 32 ], cholesterol depletion in cryopreserved sperm is deduced to happen in membrane rafts.…”

“…We recently reported in chicken sperm that membrane rafts became localized to the plasma membrane overlying the sperm head, including the APM region [ 20 ]. Furthermore, a comparison of membrane properties between fresh and cryopreserved sperm demonstrated that alteration of membrane rafts occurs concomitantly with cholesterol loss during cryopreservation, which results in impairment of post-thaw fertilizing ability [ 21 ]. These data suggest that membrane rafts play a more substantial role in regulation of sperm function during fertilization.…”

Motility, Viability and Fertilizing Ability of Avian Sperm Stored Under <i>in Vitro</i> Conditions

“…Nowadays, artificial insemination (AI) is being used widely in the breeding farm to propagate next generations. AI is often limited due to poor survival of cryopreserved avian sperm (Ushiyama et al, 2016). In this review, motility, viability and their fertilizing ability of sperm stored in vitro liquid conditions are reviewed with the knowledge of in vivo sperm survival mechanism(s).…”

Membrane-Mediated Regulation of Sperm Fertilization Potential in Poultry

“…In line with this, raft-enriched glycolipids contribute to binding the egg envelope in sea urchins (Maehashi, et al, 2003) and rainbow trout sperm (Yu, et al, 2002). Because our group found that the plasma membrane overlying the sperm head contains membrane rafts in chickens (Asano, et al, 2016), we characterized the proteome associated with sperm rafts and successfully identified 82 proteins exclusively or relatively more enriched in the membrane rafts compared to non-rafts . Furthermore, an in vitro overlay assay showed a high binding affinity for membrane raft proteins with an abundance of a 60 kDa molecule.…”