Analysis of CAPZA3 localization reveals temporally discrete events during the acrosome reaction

In mammals, the starting point of development is the fusion between sperm and egg. It is well established that sperm fuse with the egg through the equatorial/post-acrosomal region. Apart from this observation and the requirement of two proteins (CD9 in the egg and IZUMO1 in the sperm) very little is known about this fundamental process. Actin polymerization correlates with sperm capacitation in different mammalian species and it has been proposed that F-actin breakdown is needed during the acrosome reaction. Recently, we have presented evidence that actin polymerization inhibitors block the movement of IZUMO1 that accompany the acrosome reaction. These results suggest that actin dynamics play a role in the observed changes in IZUMO1 localization. This finding is significant because IZUMO1 localization in acrosome-intact sperm is not compatible with the known location of the initiation of the fusion between the sperm and the egg. To further understand the actin-mediated changes in protein localization during the acrosome reaction, the distribution of the sperm- specific plus-end actin capping protein CAPZA3 was analyzed. Like IZUMO1, CAPZA3 shows a dynamic pattern of localization; however, these movements follow a different temporal pattern than the changes observed with IZUMO1. In addition, the actin polymerization inhibitor latrunculin A was unable to alter CAPZA3 movement.Fil: Sosnik, Julian. University Of Massachussets; Estados UnidosFil: Buffone, Mariano Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina; ArgentinaFil: Visconti, Pablo E.. University Of Massachussets; Estados Unido

Central role of soluble adenylyl cyclase and cAMP in sperm physiology

Cyclic adenosine 3′,5′-monophosphate (cAMP), the first second messenger to be described, plays a central role in cell signaling in a wide variety of cell types. Over the last decades, a wide body of literature addressed the different roles of cAMP in cell physiology, mainly in response to neurotransmitters and hormones. cAMP is synthesized by a wide variety of adenylyl cyclases that can generally be grouped in two types: transmembrane adenylyl cyclase and soluble adenylyl cyclases. In particular, several aspects of sperm physiology are regulated by cAMP produced by a single atypical adenylyl cyclase (Adcy10, aka sAC, SACY). The signature that identifies sAC among other ACs, is their direct stimulation by bicarbonate. The essential nature of cAMP in sperm function has been demonstrated using gain of function as well as loss of function approaches. This review unifies state of the art knowledge of the role of cAMP and those enzymes involved in cAMP signaling pathways required for the acquisition of fertilizing capacity of mammalian sperm. This article is part of a Special Issue entitled: The role of soluble adenylyl cyclase in health and disease.Fil: Buffone, Mariano Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Wertheimer Hermitte, Eva Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Visconti, Pablo E.. University Of Massachussets; Estados UnidosFil: Krapf, Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario; Argentin

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

Bicarbonate is required for migration of sperm epididymal protein DE/CRISP-1 to the equatorial segment and expression of rat sperm fusion ability

Numerous studies have demonstrated that sperm capacitation is a bicarbonate-dependent process. In the rat, capacitation has not been studied as much as in other species, mainly because of the difficulties in carrying out functional assays with this animal model. In the present study, we have examined the influence of bicarbonate in the overall rat sperm capacitation process by analyzing involvement of the anion in 1) protein tyrosine phosphorylation, 2) migration of epididymal protein DE (also known as CRISP-1) from the dorsal region to the equatorial segment of the sperm head that occurs during capacitation, and 3) ability of sperm to fuse with the egg. Incubation of sperm under capacitating conditions produced a time-dependent increase in protein tyrosine phosphorylation. This phosphorylation did not occur in the absence of HCO3- and rapidly increased by either exposure of sperm to HCO3- or replacement of the anion by a cAMP analog (dibutyryl-cAMP) and a phosphodiesterase inhibitor (pentoxifylline). The absence of HCO3- also produced a significant decrease in the percentage of cells showing migration of DE to the equatorial segment. This parameter was completely restored by addition of the anion, but dibutyryl-cAMP and pentoxifylline were not sufficient to overcome the decrease in DE migration. Sperm capacitated in the absence of HCO3- were unable to penetrate zona-free eggs independent of the presence of the anion during gamete coincubation. Exposure of these sperm to bicarbonate, or replacement of the anion by dibutyryl-cAMP and pentoxifylline, only partially restored the sperm fusion ability. Altogether, these results indicate that, in addition to its influence on protein tyrosine phosphorylation, bicarbonate is required to support other rat sperm capacitation- associated events, such as migration of DE to the equatorial segment, and expression of the ability of sperm to fuse with the egg.Fil: Da Ros, Vanina Gabriela. 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: Munuce, María José. 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: Cohen, Debora Juana. 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: 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: Busso, Dolores. 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 UnidosFil: Cuasnicu, Patricia Sara. 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

Manipulation of bicarbonate concentration in sperm capacitation media improves in vitro fertilisation output in porcine species

BackgroundThe in vivo concentration of bicarbonate (HCO3-), one of the essential sperm capacitating effectors, varies greatly in the different environments sperm go through from cauda epididymis to the fertilisation site. On the contrary, porcine in vitro sperm capacitation and fertilisation media usually contains a standard concentration of 25mmol/L, and one of the main problems presented is the unacceptable high incidence of polyspermy. This work hypothesised that by modifying the HCO3- concentration of the medium, the output of in vitro sperm capacitation and fertilisation could be increased.ResultsOnce exposed to the capacitation medium, the intracellular pH (pH(i)) of spermatozoa increased immediately even at low concentrations of HCO3-, but only extracellular concentrations of and above 15mmol/L increased the substrates protein kinase A phosphorylation (pPKAs). Although with a significant delay, 15mmol/L of HCO3- stimulated sperm linear motility and increased other late events in capacitation such as tyrosine phosphorylation (Tyr-P) to levels similar to those obtained with 25mmol/L. This information allowed the establishment of a new in vitro fertilisation (IVF) system based on the optimization of HCO3- concentration to 15mmol/L, which led to a 25.3% increment of the viable zygotes (8.6% in the standard system vs. 33.9%).ConclusionsOptimising HCO3- concentrations allows for establishing an IVF method that significantly reduced porcine polyspermy and increased the production of viable zygotes. A concentration of 15mmol/L of HCO3- in the medium is sufficient to trigger the in vitro sperm capacitation and increase the fertilisation efficiency in porcine.This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO), the European Regional Development Fund (FEDER), Grants AGL2012-40180-C03-01-02 and AGL2015-66341-R), Fundacion Seneca (20040/GERM/16) and by a grant R01-HD-038082 (to P. E. V.) from the National Institutes of Health (NIH), USA

Ca2+ ionophore A23187 can make mouse spermatozoa capable of fertilizing in vitro without activation of cAMP-dependent phosphorylation pathways

Ca2+ ionophore A23187 is known to induce the acrosome reaction of mammalian spermatozoa, but it also quickly immobilizes them. Although mouse spermatozoa were immobilized by this ionophore, they initiated vigorous motility (hyperactivation) soon after this reagent was washed away by centrifugation. About half of live spermatozoa were acrosome-reacted at the end of 10 min of ionophore treatment; fertilization of cumulus-intact oocytes began as soon as spermatozoa recovered their motility and before the increase in protein tyrosine phosphorylation, which started 30-45 min after washing out the ionophore. When spermatozoa were treated with A23187, more than 95% of oocytes were fertilized in the constant presence of the protein kinase A inhibitor, H89. Ionophore-treated spermatozoa also fertilized 80% of oocytes, even in the absence of HCO3-, a component essential for cAMP synthesis under normal in vitro conditions. Under these conditions, fertilized oocytes developed into normal offspring. These data indicate that mouse spermatozoa treated with ionophore are able to fertilize without activation of the cAMP/PKA signaling pathway. Furthermore, they suggest that the cAMP/PKA pathway is upstream of an intracellular Ca2+ increase required for the acrosome reaction and hyperactivation of spermatozoa under normal in vitro conditions.Fil: Tateno, Hiroyuki. Asahikawa Medical University. Department of Biological Sciences; JapónFil: Krapf, Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Hino,Toshiaki. Asahikawa Medical University. Department of Biological Sciences; JapónFil: Sánchez Cárdenas, Claudia. Universidad Nacional Autonoma de Mexico. Instituto de Biotecnologia; MéxicoFil: Darszon, Alberto. Universidad Nacional Autonoma de Mexico. Instituto de Biotecnologia; MéxicoFil: Yanagimachi, Ryuzo. University of Hawaii Medical School. Institute for Biogenesis Research; Estados UnidosFil: Visconti, Pablo E.. University Of Massachussets; Estados Unido

The actin cytoskeleton of the mouse sperm flagellum is organized in a helical structure

Conception in mammals is determined by the fusion of a sperm cell with an oocyte during fertilization. Motility is one of the features of sperm that allows them to succeed in fertilization, and their flagellum is essential for this function. Longitudinally, the flagellum can be divided into the midpiece, the principal piece and the end piece. A precise cytoskeletal architecture of the sperm tail is key for the acquisition of fertilization competence. It has been proposed that the actin cytoskeleton plays essential roles in the regulation of sperm motility; however, the actin organization in sperm remains elusive. In the present work, we show that there are different types of actin structures in the sperm tail by using three-dimensional stochastic optical reconstruction microscopy (STORM). In the principal piece, actin is radially distributed between the axoneme and the plasma membrane. The actin-associated proteins spectrin and adducin are also found in these structures. Strikingly, polymerized actin in the midpiece forms a double-helix that accompanies mitochondria. Our findings illustrate a novel specialized structure of actin filaments in a mammalian cell.Fil: Gervasi, Maria Gracia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentina. University of Massachussets; Estados UnidosFil: Xu, Xinran. State University of Colorado - Fort Collins; Estados UnidosFil: Carbajal Gonzalez, Blanca. Mount Holyoke College; Estados UnidosFil: 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; ArgentinaFil: Visconti, Pablo E.. University of Massachussets; Estados UnidosFil: Krapf, Diego. State University of Colorado - Fort Collins; Estados Unido

Caput Ligation Renders Immature Mouse Sperm Motile and Capable to Undergo cAMP-Dependent Phosphorylation

Mammalian sperm must undergo two post-testicular processes to become fertilization-competent: maturation in the male epididymis and capacitation in the female reproductive tract. While caput epididymal sperm are unable to move and have not yet acquired fertilization potential, sperm in the cauda epididymis have completed their maturation, can move actively, and have gained the ability to undergo capacitation in the female tract or in vitro. Due to the impossibility of mimicking sperm maturation in vitro, the molecular pathways underlying this process remain largely unknown. We aimed to investigate the use of caput epididymal ligation as a tool for the study of sperm maturation in mice. Our results indicate that after seven days of ligation, caput sperm gained motility and underwent molecular changes comparable with those observed for cauda mature sperm. Moreover, ligated caput sperm were able to activate pathways related to sperm capacitation. Despite these changes, ligated caput sperm were unable to fertilize in vitro. Our results suggest that transit through the epididymis is not required for the acquisition of motility and some capacitation-associated signaling but is essential for full epididymal maturation. Caput epididymal ligation is a useful tool for the study of the molecular pathways involved in the acquisition of sperm motility during maturation

Manipulation of bicarbonate concentration in sperm capacitation media improves in vitro fertilisation output in porcine species

[EN] Background: The in vivo concentration of bicarbonate (HCO3), one of the essential sperm capacitating effectors, varies greatly in the different environments sperm go through from cauda epididymis to the fertilisation site. On the contrary, porcine in vitro sperm capacitation and fertilisation media usually contains a standard concentration of 25 mmol/L, and one of the main problems presented is the unacceptable high incidence of polyspermy. This work hypothesised that by modifying the HCO3 concentration of the medium, the output of in vitro sperm capacitation and fertilisation could be increased. Results: Once exposed to the capacitation medium, the intracellular pH (pHi) of spermatozoa increased immediately even at low concentrations of HCO3, but only extracellular concentrations of and above 15 mmol/L increased the substrates protein kinase A phosphorylation (pPKAs). Although with a significant delay, 15 mmol/L of HCO3 stimulated sperm linear motility and increased other late events in capacitation such as tyrosine phosphorylation (Tyr-P) to levels similar to those obtained with 25 mmol/L. This information allowed the establishment of a new in vitro fertilisation (IVF) system based on the optimization of HCO3 concentration to 15 mmol/L, which led to a 25.3% increment of the viable zygotes (8.6% in the standard system vs. 33.9%). Conclusions: Optimising HCO3 concentrations allows for establishing an IVF method that significantly reduced porcine polyspermy and increased the production of viable zygotes. A concentration of 15 mmol/L of HCO3 in the medium is sufficient to trigger the in vitro sperm capacitation and increase the fertilisation efficiency in porcineSIThis work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO), the European Regional Development Fund (FEDER), Grants AGL2012–40180-C03–01-02 and AGL2015–66341-R), Fundación Séneca (20040/GERM/16) and by a grant R01-HD-038082 (to P.E.V.) from the National In- stitutes of Health (NIH), US