MRP4-mediated cAMP efflux is essential for mouse spermatozoa capacitation.

Mammalian spermatozoa must undergo biochemical and structural changes to acquire the capacity for fertilization, in a process known as capacitation. Activation of PKA enzymes is essential for capacitation, and thus cAMP levels are tightly regulated during this process. Previously, we demonstrated that during capacitation, bovine spermatozoa extrude cAMP through multidrug resistance-associated protein 4 (MRP4, also known as ABCC4), which regulates intracellular levels of the nucleotide and provides cAMP to the extracellular space. Here, we report the presence of functional MRP4 in murine spermatozoa, since its pharmacological inhibition with MK571 decreased levels of extracellular cAMP. This also produced a sudden increase in PKA activity, with decreased tyrosine phosphorylation at the end of capacitation. Blockade of MRP4 inhibited induction of acrosome reaction, hyperactivation and in vitro fertilization. Moreover, MRP4 inhibition generated an increase in Ca2+ levels mediated by PKA, and depletion of Ca2+ salts from the medium prevented the loss of motility and phosphotyrosine inhibition produced by MK571. These results were supported using spermatozoa from CatSper Ca2+ channel knockout mice. Taken together, these results suggest that cAMP efflux via MRP4 plays an essential role in mouse sperm capacitation.This article has an associated First Person interview with the first author of the paper.

Fibroblast growth factor 2 (FGF2) is present in human spermatozoa and is related with sperm motility. The use of recombinant FGF2 to improve motile sperm recovery.

Fibroblast growth factors (FGFs) and their receptors (FGFRs) regulate several functions of somatic cells. In a previous work, we reported FGFR expression in human spermatozoa and their involvement in motility. This study aimed to evaluate the presence and localization of fibroblast growth factor 2 (FGF2) in human spermatozoa, to determine the relationship of FGF2 levels with conventional semen parameters and to assess the effect of recombinant FGF2 (rFGF2) on sperm recovery in a selection procedure. Western immunoblotting analysis using an antibody against FGF2 revealed an 18-kDa band in sperm protein extracts. The protein was immunolocalized in the sperm flagellum and acrosomal region, as well as in all germ cells. Sperm FGF2 levels, assessed by flow cytometry, showed a positive (p < 0.05) correlation with sperm concentration, motility, total sperm number and total motile cells per ejaculate. Moreover, samples with abnormal motility depicted diminished (p < 0.01) FGF2 levels compared to those with normal motility. Spermatozoa exposed to rFGF2 bound the protein, exhibited higher (p < 0.05) total and motile sperm recoveries, and increased (p < 0.01) kinematic parameters after the swim-up. Findings herein presented lead to consider sperm FGF2 level as a potential marker of sperm quality, and rFGF2 as a supplement for improving sperm recovery in selection techniques.

Molecular cloning and characterization of phospholipase C zeta in equine sperm and testis reveals species-specific differences in expression of catalytically active protein.

Oocyte activation at fertilization is brought about by the testis-specific phospholipase C zeta (PLCZ), owing to its ability to induce oscillations in intracellular Ca(2+) concentration ([Ca(2+)](i)). Whereas this is a highly conserved mechanism among mammals, important species-specific differences in PLCZ sequence, activity, and expression have been reported. Thus, the objectives of this research were to clone and characterize the intracellular Ca(2+)-releasing activity and expression of equine PLCZ in sperm and testis. Molecular cloning of equine PLCZ yielded a 1914-bp sequence that translated into a protein of the appropriate size (~73 kDa), as detected with an anti-PLCZ-specific antibody. Microinjection of 1 µg/µl of equine PLCZ cRNA supported [Ca(2+)](i) oscillations in murine oocytes that were of a higher relative frequency than those generated by an equivalent concentration of murine Plcz cRNA. Immunofluorescence revealed expression of PLCZ over the acrosome, equatorial segment, and head-midpiece junction; unexpectedly, PLCZ also localized to the principal piece of the flagellum in all epididymal, uncapacitated, and capacitated sperm. Immunostaining over the acrosome was abrogated after induction of acrosomal exocytosis. Moreover, injection of either sperm heads or tails into mouse oocytes showed that PLCZ in both fractions is catalytically active. Immunohistochemistry on equine testis revealed expression as early as the round spermatid stage, and injection of these cells supported [Ca(2+)](i) oscillations in oocytes. In summary, we report that equine PLCZ displays higher intrinsic intracellular Ca(2+)-releasing activity than murine PLCZ and that catalytically active protein is expressed in round spermatids as well as the sperm flagellum, emphasizing important species-specific differences. Moreover, some of these results may suggest potential novel roles for PLCZ in sperm physiology.

Decreased protein tyrosine phosphorylation and membrane fluidity in spermatozoa from infertile men with varicocele.

Varicocele is a prevalent pathology among infertile men. The mechanisms linking this condition to infertility, however, are poorly understood. Our previous work showed a relationship between sperm functional quality and the ability of spermatozoa to respond to capacitating conditions with increased membrane fluidity and protein tyrosine phosphorylation. Given the reported association between varicocele, oxidative stress, and sperm dysfunction, we hypothesized that spermatozoa from infertile patients with varicocele might have a combined defect at the level of membrane fluidity and protein tyrosine phosphorylation. Semen samples from infertile patients with and without grade II/III left varicocele were evaluated for motion parameters (computer-assisted semen analysis [CASA]), hyperactivation (CASA), incidence and intensity of protein tyrosine phosphorylation (phosphotyrosine immunofluorescence and western blotting), and membrane fluidity (Laurdan fluorometry), before and after a capacitating incubation (6 hr at 37 degrees C in Ham's F10/BSA, 5% CO(2)). Spermatozoa from varicocele samples presented a decreased response to the capacitating challenge, showing significantly lower motility, hyperactivation, incidence and intensity of tyrosine phosphorylation, and membrane fluidity. The findings reported in this article indicate that the sperm dysfunction associated to infertile varicocele coexists with decreased sperm plasma membrane fluidity and tyrosine phosphorylation. These deficiencies represent potential new pathophysiological mechanisms underlying varicocele-related infertility.

Human sperm subpopulations: relationship between functional quality and protein tyrosine phosphorylation.

BACKGROUND: Human semen is composed of a heterogeneous population of sperm with varying degrees of structural and functional differentiation and normality, which result in subpopulations of different quality. METHODS: Using a discontinuous Percoll gradient, we separated three subsets of sperm [(45%; L45), (65%; L65) and (90%; L90) fractions] from normozoospermic human semen samples from healthy donors and proceeded to characterize their morphology, motility and hyperactivation, as well as their ability to undergo tyrosine phosphorylation under capacitating conditions. RESULTS: As expected, sperm isolated from the lowest density layer (L45) showed the poorest quality, displaying the smallest percentage of morphologically normal and motile sperm. During a capacitating incubation, this subset of cells also showed deficient capacity to undergo hyperactivation and protein tyrosine phosphorylation. Conversely, sperm isolated from the other layers (L65 and L90) showed a time-dependent progressive increment in tyrosine phosphorylation, establishing statistically significant differences with sperm from L45. The tyrosine phosphorylation deficiency of L45 sperm could be overcome when sperm from that fraction were stimulated with activators of the cAMP-dependent kinase (PKA) pathway (dbcAMP + pentoxifylline), pointing to the sperm's plasma membrane as the main site of such deficiency. CONCLUSIONS: Poor quality sperm isolated from a Percoll gradient display an intrinsic tyrosine phosphorylation deficiency, possibly caused by a plasma membrane defect, which is associated with their inability to undergo normal capacitation and, ultimately, acquire optimal fertilizing potential.

Effects of long-term in vitro incubation of human spermatozoa: functional parameters and catalase effect.

Prolonged incubation of human spermatozoa can have deleterious effects on sperm function. The aim of this paper was to describe the effects of a prolonged in vitro incubation, under similar conditions to those employed in human assisted reproduction, on various sperm functional parameters, and to investigate the effect of an antioxidant (catalase) on this system. Freshly collected ejaculates from 20 healthy donors were studied. Samples were divided into two aliquots: the first was incubated with Ham's F10 containing 3.5% HAS, and the second was incubated in the same medium plus catalase (100 units ml-1). All experiments were carried out with spermatozoa isolated using the swim-up technique. Spermatozoa recovered from the supernatant after 1 h (T1) of incubation in 5% CO2 in air at 37 degrees C, and after 5 h (T6), 23 h (T24) and 47 h (T48), were evaluated for concentration, motion parameters including hyperactivation (computer-assisted analysis), viability, ATP concentration, reactive oxygen species (ROS) generation, DNA integrity (acridine orange), and acrosome reaction (AR). The major alteration observed in sperm function during the prolonged in vitro incubation was a reduction in the number of motile spermatozoa, together with an impairment in the quality of sperm movement. ROS levels increased with the incubation time. No substantial modifications of sperm viability, chromatin condensation and AR inducibility were observed. The addition of catalase to the medium, while keeping ROS values within baseline levels, did not prevent the loss of motility or the corresponding increase in ATP.