Bicarbonate-Stimulated Membrane Reorganization in Stallion Spermatozoa.

Paula Piccolo Maitan,Chris H Van De Lest,Tzviya Zeev-Ben-Mordehai,Bart Leemans,Tom A E Stout,Miguel Ricardo Leung,Romy Hoogendijk,Heiko Henning,Elizabeth G Bromfield,Bart M Gadella,José Domingos Guimarães,Jeroen W A Jansen

doi:10.3389/fcell.2021.772254

Abstract

Classical in vitro fertilization (IVF) is still poorly successful in horses. This lack of success is thought to be due primarily to inadequate capacitation of stallion spermatozoa under in vitro conditions. In species in which IVF is successful, bicarbonate, calcium, and albumin are considered the key components that enable a gradual reorganization of the sperm plasma membrane that allows the spermatozoa to undergo an acrosome reaction and fertilize the oocyte. The aim of this work was to comprehensively examine contributors to stallion sperm capacitation by investigating bicarbonate-induced membrane remodelling steps, and elucidating the contribution of cAMP signalling to these events. In the presence of capacitating media containing bicarbonate, a significant increase in plasma membrane fluidity was readily detected using merocyanine 540 staining in the majority of viable spermatozoa within 15 min of bicarbonate exposure. Specific inhibition of soluble adenylyl cyclase (sAC) in the presence of bicarbonate by LRE1 significantly reduced the number of viable sperm with high membrane fluidity. This suggests a vital role for sAC-mediated cAMP production in the regulation of membrane fluidity. Cryo-electron tomography of viable cells with high membrane fluidity revealed a range of membrane remodelling intermediates, including destabilized membranes and zones with close apposition of the plasma membrane and the outer acrosomal membrane. However, lipidomic analysis of equivalent viable spermatozoa with high membrane fluidity demonstrated that this phenomenon was neither accompanied by a gross change in the phospholipid composition of stallion sperm membranes nor detectable sterol efflux (p > 0.05). After an early increase in membrane fluidity, a significant and cAMP-dependent increase in viable sperm with phosphatidylserine (PS), but not phosphatidylethanolamine (PE) exposure was noted. While the events observed partly resemble findings from the in vitro capacitation of sperm from other mammalian species, the lack of cholesterol removal appears to be an equine-specific phenomenon. This research will assist in the development of a defined medium for the capacitation of stallion sperm and will facilitate progress toward a functional IVF protocol for horse gametes.

Highlights

To fertilize an oocyte, spermatozoa must undergo a process called capacitation, which begins when the spermatozoa enter the female reproductive tract, or are exposed to specific in vitro capacitation media
Our results indicated that incubating stallion spermatozoa in TyrBic medium resulted in a significantly increased population of viable spermatozoa with high membrane fluidity after a 15 min incubation time (Figure 1)
The following studies were designed to determine whether the impact of bicarbonate on stallion sperm membrane fluidity is mediated by soluble adenylyl cyclase (sAC)

Summary

Introduction

Spermatozoa must undergo a process called capacitation, which begins when the spermatozoa enter the female reproductive tract, or are exposed to specific in vitro capacitation media. Capacitation was originally defined as the physiological membrane changes that take place inside the female reproductive tract and that enable spermatozoa to acquire fertilizing capacity (Chang 1951; Austin 1952). Classical in vitro capacitation techniques (i.e., the inclusion of bicarbonate in the medium) achieve varying degrees of success in different species (Bailey, 2010). Classical IVF (gamete co-incubation) is still a very poorly successful technique that has only ever yielded two foals, both born in France in the early 1990s (Palmer et al, 1991) and live foal production has not been reproduced. The fact that in vitro matured oocytes transferred to the oviduct of an inseminated mare yield a similar percentage of embryos to spontaneous ovulation (Hinrichs et al, 2002) and that in vitro treated sperm fail to penetrate both in vivo and in vitro matured oocytes (Tremoleda et al, 2003), suggests that the deficit in equine IVF may reside in an inability to adequately induce capacitation of stallion sperm under in vitro conditions

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