RESUMEN
The acquisition of fertilization capacity by sperm is regulated by intracellular pH (pH(i)), but the transport pathways that regulate pH(i) are not well understood. Lishko et al. (2010) now report that Hv1, the voltage-sensitive proton channel, is present in human sperm and is an important regulator of the functional maturation of sperm.
Asunto(s)
Capacitación Espermática , Animales , Humanos , Concentración de Iones de Hidrógeno , Masculino , Potenciales de la Membrana , Ratones , Espermatozoides/citología , Espermatozoides/metabolismoRESUMEN
Enkurin was identified initially in mouse sperm where it was suggested to act as an intracellular adaptor protein linking membrane calcium influx to intracellular signaling pathways. In order to examine the function of this protein, a targeted mutation was introduced into the mouse Enkurin gene. Males that were homozygous for this mutated allele were subfertile. This was associated with lower rates of sperm transport in the female reproductive tract, including reduced entry into the oviduct and slower migration to the site of fertilization in the distal oviduct, and with poor progressive motility in vitro. Flagella from wild-type animals exhibited symmetrical bending and progressive motility in culture medium, and demembranated flagella exhibited the "curlicue" response to Ca2+ in vitro. In contrast, flagella of mice homozygous for the mutated allele displayed only asymmetric bending, nonprogressive motility, and a loss of Ca2+-responsiveness following demembrantion. We propose that Enkurin is part of a flagellar Ca2+-sensor that regulates bending and that the motility defects following mutation of the locus are the proximate cause of subfertility.
Asunto(s)
Proteínas de Unión a Calmodulina/fisiología , Proteínas de Plasma Seminal/fisiología , Motilidad Espermática/fisiología , Animales , Calcio/fisiología , Proteínas de Unión a Calmodulina/genética , Femenino , Infertilidad Masculina/genética , Infertilidad Masculina/fisiopatología , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Mutantes , Mutagénesis , Oviductos/citología , Oviductos/fisiología , Embarazo , Proteínas de Plasma Seminal/genética , Motilidad Espermática/genética , Cola del Espermatozoide/fisiología , Transporte Espermático/genética , Transporte Espermático/fisiologíaRESUMEN
STUDY QUESTION: Are significant abnormalities in outward (K(+)) conductance and resting membrane potential (Vm) present in the spermatozoa of patients undertaking IVF and ICSI and if so, what is their functional effect on fertilization success? SUMMARY ANSWER: Negligible outward conductance (≈5% of patients) or an enhanced inward conductance (≈4% of patients), both of which caused depolarization of Vm, were associated with a low rate of fertilization following IVF. WHAT IS KNOWN ALREADY: Sperm-specific potassium channel knockout mice are infertile with defects in sperm function, suggesting that these channels are essential for fertility. These observations suggest that malfunction of K(+) channels in human spermatozoa might contribute significantly to the occurrence of subfertility in men. However, remarkably little is known of the nature of K(+) channels in human spermatozoa or the incidence and functional consequences of K(+) channel defects. STUDY DESIGN, SIZE AND DURATION: Spermatozoa were obtained from healthy volunteer research donors and subfertile IVF and ICSI patients attending a hospital assisted reproductive techniques clinic between May 2013 and December 2015. In total, 40 IVF patients, 41 ICSI patients and 26 normozoospermic donors took part in the study. PARTICIPANTS/MATERIALS, SETTING, METHODS: Samples were examined using electrophysiology (whole-cell patch clamping). Where abnormal electrophysiological characteristics were identified, spermatozoa were further examined for Ca(2+) influx induced by progesterone and penetration into viscous media if sufficient sample was available. Full exome sequencing was performed to specifically evaluate potassium calcium-activated channel subfamily M α 1 (KCNMA1), potassium calcium-activated channel subfamily U member 1 (KCNU1) and leucine-rich repeat containing 52 (LRRC52) genes and others associated with K(+) signalling. In IVF patients, comparison with fertilization rates was done to assess the functional significance of the electrophysiological abnormalities. MAIN RESULTS AND THE ROLE OF CHANCE: Patch clamp electrophysiology was used to assess outward (K(+)) conductance and resting membrane potential (Vm) and signalling/motility assays were used to assess functional characteristics of sperm from IVF and ICSI patient samples. The mean Vm and outward membrane conductance in sperm from IVF and ICSI patients were not significantly different from those of control (donor) sperm prepared under the same conditions, but variation between individuals was significantly greater (P< 0.02) with a large number of outliers (>25%). In particular, in ≈10% of patients (7/81), we observed either a negligible outward conductance (4 patients) or an enhanced inward current (3 patients), both of which caused depolarization of Vm. Analysis of clinical data from the IVF patients showed significant association of depolarized Vm (≥0 mV) with low fertilization rate (P= 0.012). Spermatozoa with electrophysiological abnormities (conductance and Vm) responded normally to progesterone with elevation of [Ca(2+)]i and penetration of viscous medium, indicating retention of cation channel of sperm (CatSper) channel function. LIMITATIONS, REASONS FOR CAUTION: For practical, technical, ethical and logistical reasons, we could not obtain sufficient additional semen samples from men with conductance abnormalities to establish the cause of the conductance defects. Full exome sequencing was only available in two men with conductance defects. WIDER IMPLICATIONS OF THE FINDINGS: These data add significantly to the understanding of the role of ion channels in human sperm function and its impact on male fertility. Impaired potassium channel conductance (Gm) and/or Vm regulation is both common and complex in human spermatozoa and importantly is associated with impaired fertilization capacity when the Vm of cells is completely depolarized. STUDY FUNDING/COMPETING INTERESTS: The majority of the data were obtained using funding from MRC project grants (#MR/K013343/1, MR/012492/1). Additional funding was provided by NHS Tayside, TENOVUS, Chief Scientist Office NRS Fellowship and University of Abertay. The authors declare that there is no conflict of interest. TRIAL REGISTRATION NUMBER: Not applicable.
Asunto(s)
Infertilidad Masculina/genética , Potenciales de la Membrana/genética , Canales de Potasio/fisiología , Espermatozoides/química , Señalización del Calcio , Femenino , Fertilización/fisiología , Fertilización In Vitro , Humanos , Infertilidad Masculina/metabolismo , Masculino , Técnicas de Placa-Clamp , Canales de Potasio/genética , Canales de Potasio/metabolismo , Motilidad Espermática , Espermatozoides/metabolismoRESUMEN
PKDREJ is a testis-specific protein thought to be located on the sperm surface. Functional studies in the mouse revealed that loss of PKDREJ has effects on sperm transport and the ability to undergo an induced acrosome reaction. Thus, PKDREJ has been considered a potential target of post-copulatory sexual selection in the form of sperm competition. Proteins involved in reproductive processes often show accelerated evolution. In many cases, this rapid divergence is promoted by positive selection which may be driven, at least in part, by post-copulatory sexual selection. We analysed the evolution of the PKDREJ protein in primates and rodents and assessed whether PKDREJ divergence is associated with testes mass relative to body mass, which is a reliable proxy of sperm competition levels. Evidence of an association between the evolutionary rate of the PKDREJ gene and testes mass relative to body mass was not found in primates. Among rodents, evidence of positive selection was detected in the Pkdrej gene in the family Cricetidae but not in Muridae. We then assessed whether Pkdrej divergence is associated with episodes of sperm competition in these families. We detected a positive significant correlation between the evolutionary rates of Pkdrej and testes mass relative to body mass in cricetids. These findings constitute the first evidence of post-copulatory sexual selection influencing the evolution of a protein that participates in the mechanisms regulating sperm transport and the acrosome reaction, strongly suggesting that positive selection may act on these fertilization steps, leading to advantages in situations of sperm competition.
Asunto(s)
Primates/fisiología , Roedores/fisiología , Animales , Evolución Biológica , Erythrocebus patas , Gorilla gorilla , Humanos , Macaca mulatta , Macaca nemestrina , Masculino , Pan paniscus , Pan troglodytes , Pongo pygmaeus , Primates/clasificación , Primates/genética , Roedores/clasificación , Roedores/genética , Espermatozoides/metabolismo , Espermatozoides/fisiologíaRESUMEN
STUDY QUESTION: Are significant abnormalities of CatSper function present in IVF patients with normal sperm concentration and motility and if so what is their functional significance for fertilization success? SUMMARY ANSWER: Sperm with a near absence of CatSper current failed to respond to activation of CatSper by progesterone and there was fertilization failure at IVF. WHAT IS KNOWN ALREADY: In human spermatozoa, Ca(2+) influx induced by progesterone is mediated by CatSper, a sperm-specific Ca(2+) channel. A suboptimal Ca(2+) influx is significantly associated with, and more prevalent in, men with abnormal semen parameters, and is associated with reduced fertilizing capacity. However, abnormalities in CatSper current can only be assessed directly using electrophysiology. There is only one report of a CatSper-deficient man who showed no progesterone potentiated CatSper current. A CatSper 2 genetic abnormality was present but there was no information on the [Ca(2+)]i response to CatSper activation by progesterone. Additionally, the semen samples had indicating significant abnormalities (oligoasthenoteratozoospermia) multiple suboptimal functional responses in the spermatozoon. As such it cannot be concluded that impaired CatSper function alone causes infertility or that CatSper blockade is a potential safe target for contraception. STUDY DESIGN, SIZE, DURATION: Spermatozoa were obtained from donors and subfertile IVF patients attending a hospital assisted reproductive techniques clinic between January 2013 and December 2014. In total 134 IVF patients, 28 normozoospermic donors and 10 patients recalled due to a history of failed/low fertilization at IVF took part in the study. PARTICIPANTS/MATERIALS, SETTING, METHODS: Samples were primarily screened using the Ca(2+) influx induced by progesterone and, if cell number was sufficient, samples were also assessed by hyperactivation and penetration into viscous media. A defective Ca(2+) response to progesterone was defined using the 99% confidence interval from the distribution of response amplitudes in normozoospermic donors. Samples showing a defective Ca(2+) response were further examined in order to characterize the potential CatSper abnormalities. In men where there was a consistent and robust failure of calcium signalling, a direct assessment of CatSper function was performed using electrophysiology (patch clamping), and a blood sample was obtained for genetic analysis. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 101/102 (99%) IVF patients and 22/23 (96%) donors exhibited a normal Ca(2+) response. The mean (± SD) normalized peak response did not differ between donors and IVF patients (2.57 ± 0.68 [n = 34 ejaculates from 23 different donors] versus 2.66 ± 0.68 [n = 102 IVF patients], P = 0.63). In recall patients, 9/10 (90%) showed a normal Ca(2+) response. Three men were initially identified with a defective Ca(2+) influx. However, only one (Patient 1) had a defective response in repeat semen samples. Electrophysiology experiments on sperm from Patient 1 showed a near absence of CatSper current and exon screening demonstrated no mutations in the coding regions of the CatSper complex. There was no increase in penetration of viscous media when the spermatozoa were stimulated with progesterone and importantly there was failed fertilization at IVF. LIMITATIONS, REASONS FOR CAUTION: A key limitation relates to working with a specific functional parameter (Ca(2+) influx induced by progesterone) in fresh sperm samples from donors and patients that have limited viability. Therefore, for practical, technical and logistical reasons, some men (â¼ 22% of IVF patients) could not be screened. As such the incidence of significant Ca(2+) abnormalities induced by progesterone may be higher than the â¼ 1% observed here. Additionally, we used a strict definition of a defective Ca(2+) influx such that only substantial abnormalities were selected for further study. Furthermore, electrophysiology was only performed on one patient with a robust and repeatable defective calcium response. This man had negligible CatSper current but more subtle abnormalities (e.g. currents present but significantly smaller) may have been present in men with either normal or below normal Ca(2+) influx. WIDER IMPLICATIONS OF THE FINDINGS: These data add significantly to the understanding of the role of CatSper in human sperm function and its impact on male fertility. Remarkably, these findings provide the first direct evidence that CatSper is a suitable and specific target for human male contraception.
Asunto(s)
Canales de Calcio/metabolismo , Señalización del Calcio/genética , Fertilización/fisiología , Infertilidad Masculina/metabolismo , Espermatozoides/metabolismo , Adulto , Canales de Calcio/genética , Señalización del Calcio/efectos de los fármacos , Fertilización/genética , Fertilización In Vitro , Humanos , Infertilidad Masculina/genética , Masculino , Progesterona/farmacología , Recuento de Espermatozoides , Espermatozoides/efectos de los fármacosRESUMEN
The voltage-sensitive phosphoinositide phosphatases provide a mechanism to couple changes in the transmembrane electrical potential to intracellular signal transduction pathways. These proteins share a domain architecture that is conserved in deuterostomes. However, gene duplication events in primates, including humans, give rise to the paralogs TPTE and TPTE2 that retain protein domain organization but, in the case of TPTE, have lost catalytic activity. Here, we present evidence that these human proteins contain a functional voltage sensor, similar to that in nonmammalian orthologs. However, domains of these human proteins can also generate a noninactivating outward current that is not observed in zebra fish or tunicate orthologs. This outward current has the anticipated characteristics of a voltage-sensitive proton current and is due to the appearance of a single histidine residue in the S4 transmembrane segment of the voltage sensor. Histidine is observed at this position only during the eutherian radiation. Domains from both human paralogs generate proton currents. This apparent gain of proton channel function during the evolution of the TPTE protein family may account for the conservation of voltage sensor domains despite the loss of phosphatase activity in some human paralogs.
Asunto(s)
Evolución Molecular , Proteínas de la Membrana/fisiología , Fosfohidrolasa PTEN/fisiología , Secuencia de Aminoácidos , Animales , Línea Celular , Humanos , Potenciales de la Membrana/fisiología , Proteínas de la Membrana/química , Modelos Moleculares , Datos de Secuencia Molecular , Fosfohidrolasa PTEN/química , Técnicas de Placa-Clamp , Dominios y Motivos de Interacción de Proteínas , Estructura Terciaria de Proteína , Bombas de Protones/fisiología , Alineación de SecuenciaRESUMEN
Although substantial evidence exists that sperm ATP production via glycolysis is required for mammalian sperm function and male fertility, conflicting reports involving multiple species have appeared regarding the ability of individual glycolytic or mitochondrial substrates to support the physiological changes that occur during capacitation. Several mouse models with defects in the signaling pathways required for capacitation exhibit reductions in sperm ATP levels, suggesting regulatory interactions between sperm metabolism and signal transduction cascades. To better understand these interactions, we conducted quantitative studies of mouse sperm throughout a 2-h in vitro capacitation period and compared the effects of single substrates assayed under identical conditions. Multiple glycolytic and nonglycolytic substrates maintained sperm ATP levels and comparable percentages of motility, but only glucose and mannose supported hyperactivation. These monosaccharides and fructose supported the full pattern of tyrosine phosphorylation, whereas nonglycolytic substrates supported at least partial tyrosine phosphorylation. Inhibition of glycolysis impaired motility in the presence of glucose, fructose, or pyruvate but not in the presence of hydroxybutyrate. Addition of an uncoupler of oxidative phosphorylation reduced motility with pyruvate or hydroxybutyrate as substrates but unexpectedly stimulated hyperactivation with fructose. Investigating differences between glucose and fructose in more detail, we demonstrated that hyperactivation results from the active metabolism of glucose. Differences between glucose and fructose appeared to be downstream of changes in intracellular pH, which rose to comparable levels during incubation with either substrate. Sperm redox pathways were differentially affected, with higher levels of associated metabolites and reactive oxygen species generated during incubations with fructose than during incubations with glucose.
Asunto(s)
Fructosa/farmacología , Glucosa/farmacología , Manosa/farmacología , Ácido Pirúvico/farmacología , Capacitación Espermática/efectos de los fármacos , Animales , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Fructosa/metabolismo , Glucosa/metabolismo , Glucólisis/fisiología , Masculino , Manosa/metabolismo , Metabolómica , Ratones , Fosforilación , Ácido Pirúvico/metabolismo , Capacitación Espermática/fisiología , Motilidad Espermática/efectos de los fármacos , Motilidad Espermática/fisiología , Espermatozoides/efectos de los fármacos , Espermatozoides/metabolismo , Espermatozoides/fisiología , Tirosina/metabolismoRESUMEN
Pkdrej, a member of the polycystin-1 gene family, is expressed only in the male germ line. Male mice that are homozygous for a targeted mutation in the Pkdrej allele (Pkdrej(tm/tm)) are fertile in unrestricted mating trials, but exhibit lower reproductive success when competing with wild-type males in sequential mating trials and in artificial insemination of mixed-sperm populations. Following mating, sperm from Pkdrej(tm/tm) mice require >2 h longer than those of wild-type males to be detected within the egg/cumulus complex in the oviduct. Sperm from mice of both genotypes are able to capacitate in vitro. However, one of the component processes of capacitation, the ability to undergo a zona pellucida-evoked acrosome reaction, develops more slowly in sperm from Pkdrej(tm/tm) animals than in sperm from wild-type males. In contrast, a second component process of capacitation, the transition to hyperactivated flagellar motility, develops with a similar time course in both genotypes. These two behavioral consequences of capacitation, exocytotic competence and altered motility, are therefore differentially regulated. These data suggest that Pkdrej controls the timing of fertilization in vivo through effects on sperm transport and exocytotic competence and is a factor in postcopulatory sexual selection.
Asunto(s)
Receptores de Superficie Celular/fisiología , Capacitación Espermática , Acrosoma/metabolismo , Animales , Copulación , Femenino , Fertilización , Genotipo , Masculino , Preferencia en el Apareamiento Animal , Ratones , Ratones Transgénicos , Receptores de Superficie Celular/clasificación , Receptores de Superficie Celular/genética , Reproducción/genética , Capacitación Espermática/genética , Motilidad Espermática , Canales Catiónicos TRPP/clasificaciónRESUMEN
The acrosome reaction is a secretory event that must be completed by the sperm of many animal species prior to fusion with eggs. In mammals, exocytosis in triggered by ZP3, a glycoprotein component of the egg pellucida, following gamete contact. ZP3 promotes a sustained influx of Ca2+ into sperm that is necessary for the acrosome reaction. Here, we discuss the mechanism by which ZP3 generates Ca2+ entry, as well as the upstream events leading to this influx and downstream processes that couple it with exocytosis.
Asunto(s)
Reacción Acrosómica , Calcio/metabolismo , Espermatozoides/metabolismo , Animales , Canales de Calcio/metabolismo , Proteínas del Huevo/metabolismo , Exocitosis , Femenino , Glicoproteínas/metabolismo , Humanos , Masculino , Glicoproteínas de Membrana/metabolismo , Ratones , Modelos Biológicos , Receptores de Superficie Celular/metabolismo , Transducción de Señal , Zona Pelúcida/metabolismo , Glicoproteínas de la Zona PelúcidaAsunto(s)
Bicarbonatos/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fibrosis Quística/metabolismo , Fertilización/fisiología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Femenino , Humanos , Masculino , Embarazo , Capacitación Espermática/fisiología , Espermatozoides/metabolismoAsunto(s)
Factores Quimiotácticos/metabolismo , Quimiotaxis/fisiología , Erizos de Mar/fisiología , Transducción de Señal/fisiología , Espermatozoides/fisiología , Animales , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Canales Iónicos/metabolismo , Masculino , Modelos Biológicos , Óvulo/fisiología , Motilidad EspermáticaRESUMEN
Sperm of many animals must complete an exocytotic event, the acrosome reaction, in order to fuse with eggs. In mammals, acrosome reactions are triggered during sperm contact with the egg extracellular matrix, or zona pellucida, by the matrix glycoprotein ZP3. Here, we show that ZP3 stimulates production of phosphatidylinositol-(3,4,5)-triphosphate in sperm membranes. Phosphatidylinositol-3-kinase antagonists that prevent acrosome reactions and fertilization in vitro, while generation of this phosphoinositide in the absence of ZP3 triggered acrosome reactions. Downstream effectors of phosphatidylinositol-(3,4,5)-triphosphate in sperm include the protein kinases, Akt and PKCzeta. These studies outline a signal transduction pathway that plays an essential role in the early events of mammalian fertilization.
Asunto(s)
Reacción Acrosómica/fisiología , Proteínas del Huevo/metabolismo , Glicoproteínas de Membrana/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Receptores de Superficie Celular/metabolismo , Transducción de Señal/fisiología , Espermatozoides/fisiología , Animales , Femenino , Técnica del Anticuerpo Fluorescente , Immunoblotting , Lípidos/análisis , Masculino , Ratones , Glicoproteínas de la Zona PelúcidaRESUMEN
Polycystin-1 regulates a number of cellular processes through the formation of complexes with the polycystin-2 ion channel or with other signal transduction proteins. Polycystin-1 is expressed in many tissues but other members of this gene family are distributed in a more restricted fashion. PKDREJ expression has been detected only in the mammalian testis, where it is restricted to the spermatogenic lineage and retained in mature sperm. However, the functional characteristics of this protein and its role in sperm biology are not well understood. In this study it is shown that PKDREJ can modulate G protein signaling and associates with several members of the polycystin-2 family. These interactions, as well as polycystin-2 association with TRPC channels, are consistent with a role of this protein in the regulation of the acrosome reaction and in other aspects of sperm physiology.
Asunto(s)
Células Germinativas/metabolismo , Glicoproteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Receptores de Superficie Celular/metabolismo , Empalme Alternativo/genética , Secuencia de Aminoácidos , Canales de Calcio , Cationes/metabolismo , Células Cultivadas , Conductividad Eléctrica , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Humanos , Masculino , Glicoproteínas de Membrana/química , Proteínas de la Membrana , Datos de Secuencia Molecular , Unión Proteica , Canales Catiónicos TRPC/metabolismoRESUMEN
Sexual reproduction proceeds by fertilization; formation of new individuals by the union of haploid gametes. Recent reports in Cell and in Developmental Cell may provide new insights as to how this process begins and is regulated.
Asunto(s)
Caenorhabditis elegans/fisiología , Interacciones Espermatozoide-Óvulo/fisiología , Espermatozoides/fisiología , Zona Pelúcida/metabolismo , Animales , Femenino , Humanos , Masculino , Ratones , Ratones TransgénicosRESUMEN
The TRPC cation channel family has been implicated in receptor- or phospholipase C (PLC)-mediated Ca2+ entry into animal cells. These channels are present in mammalian sperm and are assigned a role in ZP3-evoked Ca2+ influx that drives acrosome reactions. However, the mechanisms controlling channel activity and coupling Ca2+ entry through these channels to cellular responses are not well understood. A yeast two-hybrid screen was carried out to identify TRPC-interacting proteins that would be candidate regulators or effectors. We identified a novel protein, enkurin, that is expressed at high levels in the testis and vomeronasal organ and at lower levels in selected other tissues. Enkurin interacts with several TRPC proteins (TRPC1, TRPC2, TRPC5, but not TRPC3) and colocalizes with these channels in sperm. Three protein-protein interaction domains were identified in enkurin: a C-terminal region is essential for channel interaction; an IQ motif binds the Ca2+ sensor, calmodulin, in a Ca2+-dependent manner; and a proline-rich N-terminal region contains predicted ligand sequences for SH3 domain proteins, including the SH3 domain of the p85 regulatory subunit of 1-phosphatidylinositol-3-kinase. We suggest that enkurin is an adaptor that functions to localize a Ca2+ sensitive signal transduction machinery in sperm to a Ca2+-permeable ion channel.