Capacitation Induces Cyclic Adenosine 3′,5′-Monophosphate-Dependent, but Apoptosis-Unrelated, Exposure of Aminophospholipids at the Apical Head Plasma Membrane of Boar Sperm Cells1

Abstract

The capacitating agent bicarbonate/CO(2) has been shown to induce profound changes in the architecture and dynamics within the sperm's plasma membrane lipid bilayer via a cAMP-dependent protein phosphorylation signaling pathway. Here we have investigated the effect of bicarbonate on surface exposure of endogenous aminophospholipids in boar spermatozoa, detecting phosphatidylserine (PS) with fluorescein-conjugated annexin V and phosphatidylethanolamine (PE) with fluorescein-conjugated streptavidin/biotinylated Ro-09-0198. Flow cytometric analyses revealed that incubation with 15 mM bicarbonate induced 30%-70% of live acrosome-intact cells to expose PE very rapidly; this exposure was closely related to a decrease in lipid packing order as detected by enhanced binding of merocyanine 540. PS exposure was detectable in the same proportion of cells, though its expression was slower. Confocal microscopy revealed that exposure of aminophospholipids in intact cells was restricted to the anterior acrosomal region of the head plasma membrane. Aminophospholipid exposure, merocyanine stainability, and a subsequent migration of cholesterol to the apical region of the head plasma membrane, were all under the control of the cAMP-dependent protein phosphorylation pathway. The close coupling of decreased lipid packing order with exposure of PE led us to conclude that bicarbonate was inducing phospholipid scrambling (i.e., collapse of asymmetric transverse distribution), and that the scrambling was a prerequisite for cholesterol relocation. There was no evidence whatever that the bicarbonate-induced scrambling was an apoptotic process. It was not accompanied by major loss of viability or by DNA degeneration or by loss of mitochondrial function, and it could not be blocked by the broad-specificity caspase inhibitors zVAD-fmk and BocD-fmk. In the absence of bicarbonate, scrambling could not be induced by the apoptotic agents UV, staurosporine, or cycloheximide. Bicarbonate-induced phospholipid scrambling thus appears to be an important and early physiological event in the capacitation process.

Flow cytometry for the assessment of animal sperm integrity and functionality: state of the art

“…Apoptosis markers have been detected in spermatozoa of many species, but the role of these markers is probably not yet elucidated completely. 49,[119][120][121][122] Changes of sperm membrane permeability have been considered as a typical event of apoptosis in many studies. Since cryopreservation, freezing and thawing cause cryodamage, and presumably even apoptotic-like changes, 123 apoptotic markers can be good tools for forecasting semen freezability and cellular damage occur during cryopreservation, albeit not necessarily indicting apoptosis really occurs.…”

“…Furthermore, it was stated that there is no reason of occurring apoptosis in spermatozoa. 122 However, it was evident that caspases were present in the restricted site for apoptosis in spermatozoa, which resulted in poor sperm quality. 131 Sperm head decondensation during fertilization During mammalian spermiogenesis, the sperm chromatin undergoes a step-by-step condensation and packaging mainly characterized by replacement of histones with protamines and the formation of S-S and…”

Cytometric solutions in veterinary andrology: Developments, advantages, and limitations

“…The bicarbonate ion stimulates a sperm-specific form of adenyl cyclase to produce enhanced levels of cyclic AMP. On one hand, the enhanced cAMP stimulates motility, possibly via phosphorylation of certain flagella proteins (42,43); on the other hand, it produces reversible changes in the membrane lipid architecture leading to exposure of phosphatidyl serine (PS) residues (44). The lipid reordering can be detected by the fluorescent probe merocyanine (45), and both changes take place rapidly in response to bicarbonate.…”

“…However, studies in human and boar have not been able to detect any key features of apoptotic action in the normal fully mature spermatozoon [e.g. (44,72)]. Strand breaks in sperm should rather be interpreted at this stage as indication of developmental aberration or stress damage.…”

“…As mentioned earlier, no firm evidence of apoptotic components in the normal mature sperm cell has thus far been produced, and although this test is adequate for detecting stress response, a more cautious interpretation is warranted for andrological applications. For example, apart from dynamic exposure as part of the capacitational process, stress might cause PS exposure as part of a necrotic process unrelated to apoptosis (44).…”