Evidence of the presence of calcium/calmodulin-dependent protein kinase IV in human sperm and its involvement in motility regulation

The mechanisms involved in the regulation of mammalian sperm motility are not well understood. Calcium ions (Ca(2+)) have been suggested to play a key role in the maintenance of motility; nevertheless, how Ca(2+) modulates this process has not yet been completely characterized. Ca(2+) can bind to calmodulin and this complex regulates the activity of multiple enzymes, including Ca(2+)/calmodulin-dependent protein kinases (CaM kinases). Results from this study confirmed that the presence of Ca(2+) in the incubation medium is essential for maintaining human sperm motility. The involvement of CaM kinases in Ca(2+) regulation of human sperm motility was evaluated using specific inhibitors (KN62 and KN93) or their inactive analogues (KN04 and KN92 respectively). Sperm incubation in the presence of KN62 or KN93 led to a progressive decrease in the percentage of motile cells; in particular, incubation with KN62 also reduced sperm motility parameters. These inhibitors did not alter sperm viability, protein tyrosine phosphorylation or the follicular fluid-induced acrosome reaction; however, KN62 decreased the total amount of ATP in human sperm. Immunological studies showed that Ca(2+)/calmodulin-dependent protein kinase IV (CaMKIV) is present and localizes to the human sperm flagellum. Moreover, CaMKIV activity increases during capacitation and is inhibited in the presence of KN62. This report is the first to demonstrate the presence of CaMKIV in mammalian sperm and suggests the involvement of this kinase in the regulation of human sperm motility.Fil: Marin Briggiler, Clara Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Jha, Kula N.. University of Virginia; Estados UnidosFil: Chertihin, Olga. University of Virginia; Estados UnidosFil: Buffone, Mariano Gabriel. Laboratorio de Estudios en Reproducción; ArgentinaFil: Herr, John C,. University of Virginia; Estados UnidosFil: Vazquez, Monica Hebe. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Visconti, Pablo E.. University of Massachussets; Estados Unido

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

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. 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.Fil: Buffone, Mariano Gabriel. Laboratorio de Estudios en Reproducción; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Doncel, G. F.. Eastern Virginia Medical School; Estados UnidosFil: Marin Briggiler, Clara Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Vazquez, Monica Hebe. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Calamera, J. C.. Laboratorio de Estudios en Reproducción; Argentin

CFTR/ENaC-dependent regulation of membrane potential during human sperm capacitation is initiated by bicarbonate uptake through NBC

To fertilize an egg, sperm must reside in the female reproductive tract to undergo several maturational changes that are collectively referred to as capacitation. From a molecular point of view, the HCO3-dependent activation of the atypical soluble adenylyl cyclase (ADCY10) is one of the first events that occurs during capacitation and leads to the subsequent cAMP-dependent activation of protein kinase A (PKA). Capacitation is also accompanied by hyperpolarization of the sperm plasma membrane. We previously reported that PKA activation is necessary for CFTR (cystic fibrosis transmembrane conductance regulator channel) activity and for the modulation of membrane potential (Em). However, the main HCO3 transporters involved in the initial transport and the PKA-dependent Em changes are not well known nor characterized. Here, we analyzed how the activity of CFTR regulates Em during capacitation and examined its relationship with an electrogenic Na/HCO3 cotransporter (NBC) and epithelial Na channels (ENaCs). We observed that inhibition of both CFTR and NBC decreased HCO3 influx, resulting in lower PKA activity, and that events downstream of the cAMP activation of PKA are essential for the regulation of Em. Addition of a permeable cAMP analog partially rescued the inhibitory effects caused by these inhibitors. HCO3 also produced a rapid membrane hyperpolarization mediated by ENaC channels, which contribute to the regulation of Em during capacitation. Altogether, we demonstrate for the first time, that NBC cotransporters and ENaC channels are essential in the CFTR-dependent activation of the cAMP/PKA signaling pathway and Em regulation during human sperm capacitation.Fil: Puga Molina, Lis del Carmen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Pinto, Nicolás Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Torres, Nicolás I.. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: González Cota, Ana, L.. University of Washington; Estados UnidosFil: Luque, Guillermina Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Balestrini, Paula Ania. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Romarowski, Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Krapf, Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Santi, Celia M.. University of Washington; Estados UnidosFil: Treviño, Claudia L.. Universidad Nacional Autónoma de México; MéxicoFil: Darszon, Alberto. Universidad Nacional Autónoma de México; MéxicoFil: Buffone, Mariano Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentin

Molecular Basis of Human Sperm Capacitation

In the early 1950s, Austin and Chang independently described the changes that are required for the sperm to fertilize oocytes in vivo. These changes were originally grouped under name of “capacitation” and were the first step in the development of in vitro fertilization (IVF) in humans. Following these initial and fundamental findings, a remarkable number of observations led to characterization of the molecular steps behind this process. The discovery of certain sperm-specific molecules and the possibility to record ion currents through patch-clamp approaches helped to integrate the initial biochemical observation with the activity of ion channels. This is of particular importance in the male gamete due to the fact that sperm are transcriptionally inactive. Therefore, sperm must control all these changes that occur during their transit through the male and female reproductive tracts by complex signaling cascades that include post-translational modifications. This review is focused on the principal molecular mechanisms that govern human sperm capacitation with particular emphasis on comparing all the reported pieces of evidence with the mouse model

Hypercholesterolemia Impaired Sperm Functionality in Rabbits

Hypercholesterolemia represents a high risk factor for frequent diseases and it has also been associated with poor semen quality that may lead to male infertility. The aim of this study was to analyze semen and sperm function in diet-induced hypercholesterolemic rabbits. Twelve adult White New Zealand male rabbits were fed ad libitum a control diet or a diet supplemented with 0.05% cholesterol. Rabbits under cholesterol-enriched diet significantly increased total cholesterol level in the serum. Semen examination revealed a significant reduction in semen volume and sperm motility in hypercholesterolemic rabbits (HCR). Sperm cell morphology was seriously affected, displaying primarily a “folded head”-head fold along the major axe-, and the presence of cytoplasmic droplet on sperm flagellum. Cholesterol was particularly increased in acrosomal region when detected by filipin probe. The rise in cholesterol concentration in sperm cells was determined quantitatively by Gas chromatographic-mass spectrometric analyses. We also found a reduction of protein tyrosine phosphorylation in sperm incubated under capacitating conditions from HCR. Interestingly, the addition of Protein Kinase A pathway activators -dibutyryl-cyclic AMP and iso-butylmethylxanthine- to the medium restored sperm capacitation. Finally, it was also reported a significant decrease in the percentage of reacted sperm in the presence of progesterone. In conclusion, our data showed that diet-induced hypercholesterolemia adversely affects semen quality and sperm motility, capacitation and acrosomal reaction in rabbits; probably due to an increase in cellular cholesterol content that alters membrane related events

The Tissue-Specific Rep8/UBXD6 Tethers p97 to the Endoplasmic Reticulum Membrane for Degradation of Misfolded Proteins

The protein known as p97 or VCP in mammals and Cdc48 in yeast is a versatile ATPase complex involved in several biological functions including membrane fusion, protein folding, and activation of membrane-bound transcription factors. In addition, p97 plays a central role in degradation of misfolded secretory proteins via the ER-associated degradation pathway. This functional diversity of p97 depends on its association with various cofactors, and to further our understanding of p97 function it is important that these cofactors are identified and analyzed. Here, we isolate and characterize the human protein named Rep8 or Ubxd6 as a new cofactor of p97. Mouse Rep8 is highly tissue-specific and abundant in gonads. In testes, Rep8 is expressed in post-meiotic round spermatids, whereas in ovaries Rep8 is expressed in granulosa cells. Rep8 associates directly with p97 via its UBX domain. We show that Rep8 is a transmembrane protein that localizes to the ER membrane with its UBX domain facing the cytoplasm. Knock-down of Rep8 expression in human cells leads to a decreased association of p97 with the ER membrane and concomitantly a retarded degradation of misfolded ER-derived proteasome substrates. Thus, Rep8 tethers p97 to the ER membrane for efficient ER-associated degradation

Otitis Media in a New Mouse Model for CHARGE Syndrome with a Deletion in the Chd7 Gene

Otitis media is a middle ear disease common in children under three years old. Otitis media can occur in normal individuals with no other symptoms or syndromes, but it is often seen in individuals clinically diagnosed with genetic diseases such as CHARGE syndrome, a complex genetic disease caused by mutation in the Chd7 gene and characterized by multiple birth defects. Although otitis media is common in human CHARGE syndrome patients, it has not been reported in mouse models of CHARGE syndrome. In this study, we report a mouse model with a spontaneous deletion mutation in the Chd7 gene and with chronic otitis media of early onset age accompanied by hearing loss. These mice also exhibit morphological alteration in the Eustachian tubes, dysregulation of epithelial proliferation, and decreased density of middle ear cilia. Gene expression profiling revealed up-regulation of Muc5ac, Muc5b and Tgf-β1 transcripts, the products of which are involved in mucin production and TGF pathway regulation. This is the first mouse model of CHARGE syndrome reported to show otitis media with effusion and it will be valuable for studying the etiology of otitis media and other symptoms in CHARGE syndrome

Mutation in Archain 1, a Subunit of COPI Coatomer Complex, Causes Diluted Coat Color and Purkinje Cell Degeneration

Intracellular trafficking is critical for delivering molecules and organelles to their proper destinations to carry out normal cellular functions. Disruption of intracellular trafficking has been implicated in the pathogenesis of various neurodegenerative disorders. In addition, a number of genes involved in vesicle/organelle trafficking are also essential for pigmentation, and loss of those genes is often associated with mouse coat-color dilution and human hypopigmentary disorders. Hence, we postulated that screening for mouse mutants with both neurological defects and coat-color dilution will help identify additional factors associated with intracellular trafficking in neuronal cells. In this study, we characterized a mouse mutant with a unique N-ethyl-N-nitrosourea (ENU)–induced mutation, named nur17. nur17 mutant mice exhibit both coat-color dilution and ataxia due to Purkinje cell degeneration in the cerebellum. By positional cloning, we identified that the nur17 mouse carries a T-to-C missense mutation in archain 1 (Arcn1) gene which encodes the δ subunit of the coat protein I (COPI) complex required for intracellular trafficking. Consistent with this function, we found that intracellular trafficking is disrupted in nur17 melanocytes. Moreover, the nur17 mutation leads to common characteristics of neurodegenerative disorders such as abnormal protein accumulation, ER stress, and neurofibrillary tangles. Our study documents for the first time the physiological consequences of the impairment of the ARCN1 function in the whole animal and demonstrates a direct association between ARCN1 and neurodegeneration