Effects of Semen Processing on Sperm Function: Differences between Swim-Up and Density Gradient Centrifugation.

PURPOSE: Andrology research has evolved notoriously in the latest years, particularly since male factor contribution to couple infertility has been undoubtedly demonstrated. However, sperm function investigations results are sometimes contradictory, probably as a result of the use of different sperm processing techniques. In this work, we underwent a systematic functional comparison of human sperm samples simultaneously processed by swim-up and density gradient centrifugation, which are the preferred sperm processing methods used in basic and clinical laboratories. MATERIALS AND METHODS: To compare functional characteristics of sperm isolated by swim-up and density gradient centrifugation followed by incubation at different times under capacitating conditions. RESULTS: Semen samples processed in parallel by these two procedures resulted in sperm preparations with significant differences in redox state, spontaneous intracellular calcium oscillations, hyperactivation, protein tyrosine phosphorylation, and acrosome reaction responsivity to calcium ionophore. Such differences showed time-dependent specific patterns for spontaneous intracellular calcium oscillations, hyperactivation and protein tyrosine phosphorylation. Sperm retrieved by density gradient centrifugation showed more hyperactivation and tyrosine phosphorylation than swim-up sperm, suggesting a higher degree of capacitation. CONCLUSIONS: Our results account for functional differences observed in spermatozoa processed with these two methods and therefore may contribute to a better interpretation of outcomes obtained in different laboratories as well as to improve experimental designs aimed to study sperm physiology and fertility potential.

Proteomic characterization of human sperm plasma membrane-associated proteins and their role in capacitation.

BACKGROUND: Plasma membranes of ejaculated sperm are covered by epididymal and accessory glands secreted proteins that must be released from sperm surface during the female reproductive tract passage in order to capacitate and fertilize the oocyte. OBJECTIVES: As human sperm plasma membrane-associated proteins (SMAP) have not yet been investigated, the aim of this study was to characterize the SMAP released during in vitro human capacitation and to study their possible role as decapacitation factors. MATERIALS AND METHODS: SMAP were characterized by 2-dimensional electrophoresis and mass spectrometry analysis. Besides, we explored SMAP effects on motility, protein tyrosine phosphorylation, and calcium ionophore-induced acrosome reaction of spermatozoa either incubated for 6 h in capacitating medium ± SMAP or for 5 h in capacitating medium alone followed by incubation for 1 h ± SMAP. RESULTS: Mass spectrometry analysis allowed the identification of 29 proteins, all of which have previously been identified in the human seminal fluid. Spermatozoa incubated for 6 h under capacitating conditions in the presence of the SMAP showed a significant decrease in the incidence of non-progressive motility, hyperactivation, protein tyrosine phosphorylation, and calcium ionophore-induced acrosome reaction. However, spermatozoa incubated for 5 h in capacitating medium and further incubated for 1 h with the SMAP showed a lower percentage of spermatozoa with non-progressive motility and hyperactivated cells but no effects on protein tyrosine phosphorylation were detected. DISCUSSION AND CONCLUSIONS: Our results indicate that SMAP inhibit the progress of human sperm capacitation, but only motility changes related to capacitation may be reversed by these proteins. The study of the identified proteins on sperm function and their mechanisms of action on this cell may contribute to the understanding of their role during capacitation.

Single-cell microinjection assay indicates that 7-hydroxycoumarin induces rapid activation of caspase-3 in A549 cancer cells.

Coumarins have attracted intense interest in recent years due to their apoptogenic effects. The aim of the present study was to determine whether 7-hydroxycoumarin (7-HC) induces changes in caspase-3 (C-3) activity in A549 human lung carcinoma cells. A range of analytical techniques, including colorimetric and fluorometric assays, western blotting, single-cell microinjection, fluorescence microscopy and image analysis were conducted to elucidate the effects of 7-HC. A 24-h exposure to 1.85 mM 7-HC induced a 65% increase in C-3 activity, and a notable conversion of procaspase-3 to C-3, in addition to poly(ADP-ribose)polymerase cleavage. Furthermore, morphological changes associated with apoptosis were observed. Exposure of the cells to 7-HC for 3 or 6 h increased calcium conductance by 27%. By performing the single-cell microinjection of a specific fluorescent substrate of C-3 into previously 7-HC-exposed cells, a typical enzymatic kinetic profile of C-3 activation was identified a number of hours prior to the morphological and biochemical changes associated with apoptosis being observed. These results suggest that the rapid in vivo activation of C-3 is induced by 7-HC, the most relevant biotransformation product of coumarin in humans.

Sodium influx induced by external calcium chelation decreases human sperm motility.

BACKGROUND: Calcium removal from the medium promptly reduces human sperm motility and induces a Na(+)-dependent depolarization that is accompanied by an increase in intracellular sodium concentration ([Na(+)](i)) and a decrease in intracellular calcium concentration ([Ca(2+)](i)). Sodium loading activates a Na(+)/K(+)-ATPase. METHODS: Membrane potential (Vm) and [Ca(2+)](i) were simultaneously detected in human sperm populations with the fluorescent probes diSC(3)(5) and fura 2. [Na(+)](i) and was measured independently in a similar fashion using sodium-binding benzofuran isophthalate. Motility was determined in a CASA system, ATP was measured using the luciferin-luciferase assay, and cAMP was measured by radioimmunoassay. RESULTS: Human sperm motility reduction after calcium removal is related to either Na(+)-loading or Na(+)-dependent depolarization, because, under conditions that inhibit the calcium removal-induced Na(+)-dependent depolarization and [Na(+)](i) increase, sperm motility was unaffected. By clamping sperm Vm with valinomycin, we found that the motility reduction associated with the calcium removal was related to sodium loading, and not to membrane potential depolarization. Mibefradil, a calcium channel blocker, markedly inhibited the Na(+)-dependent depolarization and sodium loading, and also preserved sperm motility. In the absence of calcium, both ATP and cAMP concentrations were decreased by 40%. However ATP levels were unchanged when calcium removal was performed under conditions that inhibit the calcium removal-induced Na(+)-dependent depolarization and [Na(+)](i) increase. CONCLUSIONS: Human sperm motility arrest induced by external calcium removal is mediated principally by sodium loading, which would stimulate the Na(+)/K(+)-ATPase and in turn deplete the ATP content.

Activation of protein kinase A stimulates the progesterone-induced calcium influx in human sperm exposed to the phosphodiesterase inhibitor papaverine.

Progesterone induces a fast transient calcium influx in human sperm though the activation of nongenomic receptors. During sperm capacitation, a complex process required for sperm to be able to fertilize the egg, the calcium influx induced by progesterone is enhanced. Sperm capacitation is mediated by an increase in cAMP content and subsequent protein kinase A (PKA) activation. In this work, we examined the effect of increasing intracellular cAMP on the calcium influx induced by progesterone in noncapacitated human sperm. To do this, sperm were exposed to the phosphodiesterase inhibitor papaverine for 5 minutes, a treatment that increased both the cAMP content and the PKA activity several-fold. The calcium influx induced by progesterone was increased by papaverine to levels close to those found in capacitated sperm. This effect was partially inhibited by H89 (48%) and by genistein (41%), and the sum of both inhibitors reduced the stimulating effect of papaverine by 89%. The inhibitory effect of genistein on the progesterone-induced calcium influx could be related to its capability to inhibit the papaverine-stimulated increase in cAMP content and PKA activity. The results presented here suggest that the calcium influx induced by progesterone is up-regulated by the PKA activity.

Intracellular sodium increase induced by external calcium removal in human sperm.

In human sperm, removal of external calcium produces a fast Na(+)-dependent depolarization that is presumably due to sodium permeation through calcium channels. Calcium restoration produces a ouabain-sensitive hyperpolarization that brings the membrane potential to values frequently more negative than resting. In this work, we show evidence indicating that external calcium removal induces an increase in the intracellular sodium ([Na(+)](i)) and that this phenomenon is related to the Na(+)-dependent depolarization. Calcium restoration blocked the [Na(+)](i) increase and then produced a slow decrease that was inhibited by ouabain. The [Na(+)](i) increase was inhibited by nanomolar-micromolar calcium or by millimolar magnesium, which has been previously shown to inhibit the Na(+)-dependent depolarization. This evidence supports the hypothesis that, in zero-calcium medium, a calcium channel that would contribute to resting intracellular calcium levels allows sodium permeation, producing depolarization and a significant [Na(+)](i) increase. Sodium loading would stimulate the Na(+),K(+)-ATPase, the activity of which contributes to the sperm hyperpolarization observed upon calcium restoration.

A remarkable increase in the pHi sensitivity of voltage-dependent calcium channels occurs in human sperm incubated in capacitating conditions.

Human sperm are endowed with voltage-dependent calcium channels (VDCC) that produce increases in [Ca2+]i in response to depolarization with KCl. These channels are stimulated during "capacitation", a complex biochemical process, accompanied by a slight pHi alkalization, that sperm must accomplish to acquire the ability to fertilize the egg. The stimulation can be explained in part by the fact that in non-capacitated sperm, calcium influx through VDCC is stimulated by pHi alkalization in the range of pHi observed during capacitation. In this work, we explored the effect of pHi on VDCC in capacitated sperm loaded with fura ff. Strikingly, the pHi sensitivity of VDCC increased approximately 7-fold when sperm was capacitated, as compared with non-capacitated sperm. This finding suggests that the pHi sensitivity of VDCC can be modulated during capacitation so that a combined effect of pHi alkalization and biochemical regulation enhances calcium influx through these channels.