Flow-cytometric analysis of membrane integrity of stallion sperm in the face of agglutination: the “zombie sperm” dilemma

Abstract

Purpose To define the effect of sperm agglutination, associated with incubation under capacitating conditions, on accuracy of membrane assessment via flow cytometry and to develop methods to mitigate that effect. Methods Sperm motility was measured by CASA. Sperm were stained with PI-PSA or a novel method, LD-PSA, using fixable live/dead stain and cell dissociation treatment, before flow-cytometric analysis. Using LD-PSA, acrosome reaction and plasma membrane status were determined in equine sperm treated with 10 μm A23187 for 10 min, followed by 0, 1, or 2 h incubation in capacitating conditions. Results Using PI-PSA, measured membrane integrity (MI; live sperm) was dramatically lower than was total motility (TMOT), indicating spurious results (“zombie sperm”). Sperm aggregates were largely of motile sperm. Loss of motility after A23187 treatment was associated with disaggregation and increased MI. On disaggregation using LD-PSA, MI rose, and MI then corresponded with TMOT. In equine sperm incubated after A23187 treatment, as the percentage of live acrosome-reacted sperm increased, TMOT decreased to near 0. Conclusion Flow cytometry assesses only individualized sperm; thus, agglutination of viable sperm alters recorded membrane integrity. As viable sperm become immotile, they individualize; therefore, factors that decrease motility, such as A23187, result in increased measured MI. Disaggregation before assessment allows more accurate determination of sperm membrane status; in this case we documented a mismatch between motility and live acrosome-reacted equine sperm that may relate to the poor repeatability of A23187 treatment for equine IVF. These findings are of profound value to future studies on sperm capacitation.

Factors affecting intracellular calcium influx in response to calcium ionophore A23187 in equine sperm

“…In previous reports using A23187 treatment to prepare mouse spermatozoa for IVF, treatment with 20 µM A23187 for 10 min in the presence of 4 mg/ml BSA in a medium with a high calcium concentration (3.42 mM) caused spermatozoa to become immotile, and then withdrawal of A23187 (by centrifugation and resuspension of spermatozoa in BSA‐containing medium) was followed within 5 min by recovery of motility, transforming within 30 min to hyperactivated motility concomitant with induction of the acrosome reaction, and gain of fertilizing ability 6 . In contrast, treatment of equine spermatozoa with 5–10 µM A23187 for 6–10 min followed by ionophore washout has been associated with a continued, ongoing decrease in motility, 31,37 falling to essentially zero motility at 2 h post‐treatment, the time at which the first significant increase in the acrosome reaction was seen 31 . The apparent inefficiency of equine spermatozoa in eliminating intracellular calcium after A23187 treatment may explain the observed inability of equine spermatozoa to recover effective motility after brief ionophore treatment, as equine sperm motility is inhibited at relatively low levels of intracellular calcium 38 .…”

“…6 In contrast, treatment of equine spermatozoa with 5-10 µM A23187 for 6-10 min followed by ionophore washout has been associated with a continued, ongoing decrease in motility, 31,37 falling to essentially zero motility at 2 h post-treatment, the time at which the first significant increase in the acrosome reaction was seen. 31 The apparent inefficiency of equine spermatozoa in eliminating intracellular calcium after A23187 treatment may explain the observed inability of equine spermatozoa to recover effective motility after brief ionophore treatment, as equine sperm motility is inhibited at relatively low levels of intracellular calcium. 38 Little information is available on the effect of A23187 treatment on important parameters of sperm function related to fertilization, that is, motility and acrosomal responsiveness in viable spermatozoa.…”

“…[2][3][4][5] While we did not determine the status of the acrosome in the spermatozoa in these studies, there is indication from previous work that appreciable changes in the proportion of acrosome reaction in fresh equine spermatozoa do not occur until 2 h after exposure to A23187 either continuously, as in this study, 29,30 or for a brief period followed by washing and incubation. 31 The increased final fluorescence found after A23187 in proteincontaining medium also suggests that the plateau in fluorescence for the different treatments is indicative of a steady-state sperm [Ca 2+ ] i for that treatment rather than to the limit of Fluo-4 saturation.…”

“…In a recent study, we found that treatment of equine spermatozoa with A23187 for 10 min under conditions similar to those in the present studies (HBSS base medium; A23187 added to sperm suspension as an HBSS-2° stock solution; 7 mg/ml fatty acid-free BSA) was associated with a significant increase in viable, acrosome-reacted spermatozoa at 2 h after treatment with 5 or 10 µM, but not 1 µM, A23187; however, sperm motility in the effective treatments was profoundly decreased, to less than 10% total motile spermatozoa, by 2 h after treatment. 31 In evaluating reasons for the difference in efficiency of murine and equine spermatozoa in normalizing [Ca 2+ ] i after A23187 removal from the medium, it is possible that the sperm membrane interacts with the A23187 molecule differently between species. It has been postulated that carboxylic ionophores are incorporated into membranes, being lipid-soluble when complexed with a cation (Ca 2+ or H + ), and then are trapped at the membrane's polar surface when the cation is released and the ionophore becomes polarized.…”

“…However, the results are controversial. For example, some researchers have reported that SARS-CoV-2 was not detected in the male reproductive tract, [25][26][27][28][29][30][31][32][33][34] while others reported that SARS-CoV-2 RNA was found in the semen or testes of COVID-19 patients. 35,36 There are also unknown factors regarding COVID-19 and male reproduction.…”