RESUMO
After ejaculation, mammalian sperm undergo a series of molecular events conducive to the acquisition of fertilizing competence. These events are collectively known as capacitation and involve acrosomal responsiveness and a vigorous sperm motility called hyperactivation. When mimicked in the laboratory, capacitating bovine sperm medium contains bicarbonate, calcium, albumin and heparin, among other components. In this study, we aimed at establishing a new capacitation protocol for bovine sperm, using calcium ionophore. Similar to our findings using mouse sperm, bovine sperm treated with Ca2+ ionophore A23187 were quickly immobilized. However, these sperm initiated capacitation after ionophore removal in fresh medium without heparin, and independent of the Protein Kinase A. When A23187-treated sperm were used on in vitro fertilization (IVF) procedures without heparin, eggs showed cleavage rates similar to standardized IVF protocols using heparin containg synthetic oviduct fluid (IVF-SOF). However, when A23187 pre-treated sperm were further used for inseminating eggs in complete IVF-SOF-heparin, a significantly higher percentage of embryo development was observed, suggesting a synergism between two different signaling pathways during bovine sperm capacitation. These results have the potential to improve current protocols for bovine IVF that could also be applied in other species of commercial interest.
Assuntos
Calcimicina , Ionóforos de Cálcio , Criopreservação , Fertilização in vitro , Preservação do Sêmen , Capacitação Espermática , Espermatozoides , Animais , Bovinos , Masculino , Ionóforos de Cálcio/farmacologia , Criopreservação/veterinária , Criopreservação/métodos , Fertilização in vitro/veterinária , Fertilização in vitro/métodos , Calcimicina/farmacologia , Espermatozoides/efeitos dos fármacos , Espermatozoides/fisiologia , Capacitação Espermática/efeitos dos fármacos , Preservação do Sêmen/veterinária , Preservação do Sêmen/métodos , Feminino , Técnicas de Cultura Embrionária/veterinária , Desenvolvimento Embrionário/efeitos dos fármacosRESUMO
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.
Assuntos
AMP Cíclico/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Capacitação Espermática/fisiologia , Animais , Cálcio/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Fosfotirosina/metabolismo , Propionatos/farmacologia , Quinolinas/farmacologia , Capacitação Espermática/efeitos dos fármacos , Motilidade dos Espermatozoides/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismoRESUMO
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.
Assuntos
Fator 2 de Crescimento de Fibroblastos/isolamento & purificação , Motilidade dos Espermatozoides/fisiologia , Espermatozoides/química , Western Blotting , Fator 2 de Crescimento de Fibroblastos/fisiologia , Citometria de Fluxo , Humanos , Masculino , Proteínas Recombinantes/farmacologia , Sêmen/química , Motilidade dos Espermatozoides/efeitos dos fármacos , Recuperação Espermática , Espermatozoides/fisiologiaRESUMO
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.
Assuntos
Infertilidade Masculina/etiologia , Fluidez de Membrana/fisiologia , Proteínas Tirosina Quinases/metabolismo , Espermatozoides/metabolismo , Varicocele/complicações , Adulto , Membrana Celular/fisiologia , Humanos , Masculino , Fosforilação , Motilidade dos EspermatozoidesRESUMO
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.
Assuntos
Espermatozoides/citologia , Espermatozoides/fisiologia , Tirosina/metabolismo , Bucladesina/farmacologia , Centrifugação com Gradiente de Concentração , Coloides , Humanos , Masculino , Movimento (Física) , Pentoxifilina/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Fosforilação/efeitos dos fármacos , Povidona , Valores de Referência , Dióxido de Silício , Capacitação Espermática , Motilidade dos Espermatozoides , Espermatozoides/metabolismoRESUMO
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.