Capacitation in the presence of methyl-β-cyclodextrin results in enhanced zona pellucida-binding ability of stallion spermatozoa

Abstract

While IVF has been widely successful in many domesticated species, the development of a robust IVF system for the horse remains an elusive and highly valued goal. A major impediment to the development of equine IVF is the fact that optimised conditions for the capacitation of equine spermatozoa are yet to be developed. Conversely, it is known that stallion spermatozoa are particularly susceptible to damage arising as a consequence of capacitation-like changes induced prematurely in response to semen handling and transport conditions. To address these limitations, this study sought to develop an effective system to both suppress and promote the in vitro capacitation of stallion spermatozoa. Our data indicated that the latter could be achieved in a bicarbonate-rich medium supplemented with a phosphodiesterase inhibitor, a cyclic AMP analogue, and methyl-b-cyclodextrin, an efficient cholesterolwithdrawing agent. The populations of spermatozoa generated under these conditions displayed a number of hallmarks of capacitation, including elevated levels of tyrosine phosphorylation, a reorganisation of the plasma membrane leading to lipid raft coalescence in the peri-acrosomal region of the sperm head, and a dramatic increase in their ability to interact with heterologous bovine zona pellucida (ZP) and undergo agonist-induced acrosomal exocytosis. Furthermore, this functional transformation was effectively suppressed in media devoid of bicarbonate. Collectively, these results highlight the importance of efficient cholesterol removal in priming stallion spermatozoa for ZP binding in vitro.

Effect of calcium, bicarbonate, and albumin on capacitation-related events in equine sperm

“…The only treatment that consistently induced a significant decrease in sperm cholesterol content was MW with MbCD at 1 and 3 mM (Figs 1 and 2). Incubation with MbCD has been shown to extract cholesterol from sperm plasmalemma of different mammalian species (Visconti et al 1999c, Bromfield et al 2014. Consistent with our findings, Boerke et al (2013) found in porcine sperm that cholesterol efflux mediated by MbCD is ROS independent, unlike BSAmediated cholesterol extraction, which requires the Figure 8 Number of equine sperm bound to bovine ZP.…”

“…An increase in PYassociated with treatment with MbCD was reported in equine sperm, but this was examined only in media containing activators of the cAMP-PKA pathway (Pommer et al 2003); such activators essentially bypass the dependence of PY on cholesterol efflux (Visconti et al 1995). Similarly, Bromfield et al (2014) have recently evaluated factors affecting cholesterol reorganization patterns and PY in equine sperm, but essentially only in the presence of pentoxifylline and dibutyryl cAMP. Incubation of equine sperm with MbCD in the basal medium (not containing stimulators of the cAMP-PKA pathway) promoted changes in measures of membrane fluidity and cholesterol organization, but did not significantly increase PY; remarkably, the effects of BSA on these parameters in the basal medium were not reported (Bromfield et al 2014).…”

“…Similarly, Bromfield et al (2014) have recently evaluated factors affecting cholesterol reorganization patterns and PY in equine sperm, but essentially only in the presence of pentoxifylline and dibutyryl cAMP. Incubation of equine sperm with MbCD in the basal medium (not containing stimulators of the cAMP-PKA pathway) promoted changes in measures of membrane fluidity and cholesterol organization, but did not significantly increase PY; remarkably, the effects of BSA on these parameters in the basal medium were not reported (Bromfield et al 2014).…”

Penicillamine prevents ram sperm agglutination in media that support capacitation

“…These findings were extended in utero by Brown & Senger (1982) who placed rabbit spermatozoa into the uterine lumen and noted significant agglutination of motile spermatozoa. Similar effects have been reported in the stallion with the use of BSA under capacitating conditions, so it is commonly substituted with polyvinyl alcohol (PVA; Bromfield et al 2014).…”

“…the stallion; (Bromfield et al 2014)). There are considerable differences in sperm morphology and physiology across mammalian species, so it is not surprising that distinct species-specific capacitation phenomena also occur.…”