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

Recruitment of Orc6l, a dormant maternal mRNA in mouse oocytes, is essential for DNA replication in 1-cell embryos

Mouse oocytes acquire the ability to replicate DNA during meiotic maturation, presumably to ensure that DNA replication does not occur precociously between MI and MII and only after fertilization. Acquisition of DNA replication competence requires protein synthesis, but the identity of the proteins required for DNA replication is poorly described. In Xenopus, the only component missing for DNA replication competence is CDC6, which is synthesized from a dormant maternal mRNA recruited during oocyte maturation, and a similar situation also occurs during mouse oocyte maturation. We report that ORC6L is another component required for acquisition of DNA replication competence that is absent in mouse oocytes. The dormant maternal Orc6l mRNA is recruited during maturation via a CPE present in its 3␣ UTR. RNAi-mediated ablation of maternal Orc6l mRNA prevents the maturation-associated increase in ORC6L protein and inhibits DNA replication in 1-cell embryos. These results suggest that mammalian oocytes have more complex mecha- nisms to establish DNA replication competence when compared to their Xenopus counterparts.Fil: Murai, Shin. State University Of Pennsylvania; Estados UnidosFil: Stein, Paula. University of Pennsylvania; Estados UnidosFil: Buffone, Mariano Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina; Argentina. University of Pennsylvania; Estados UnidosFil: Yamashita, Shigeru. Toho University. School of Medicine; JapónFil: Schultz, Richard M.. University of Pennsylvania; Estados Unido

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

AMH sérico pode predizer o risco de cancelamento de ciclo e as chances de boa resposta ovariana, independentemente da idade ou FSH

In the recent years, anti-Müllerian hormone (AMH) has been shown to represent a reliable marker of the ovarian reserve. In this study, we evaluate the risk of cycle cancellation and the chances of good ovarian response to controlled hyperstimulation and of pregnancy according to serum AMH measured prior to assisted reproduction procedures in females undergoing intracytoplasmic sperm injection. An analytic observational study included females undergoing ICSI between in a single center. Subgroup analyses were performed by grouping patients according to FSH levels or to their age. The risk of cycle cancellation decreased from 64% in patients with serum AMH ≤3 pmol/L (0.42 ng/mL) to 21% with AMH ≥15 pmol/L (2.10 ng/mL). The rate of good response increased from almost null in patients with AMH ≤3 pmol/L to 61% in those with AMH ≥15 pmol/L. Pregnancy rate increased moderately, but significantly, from 31% with AMH ≤3 pmol/L to 35% with AMH ≥15 pmol/L. Here we provide estimates of those outcomes according to the values of serum AMH, in general and in subgroups according to patient’s age or serum FSH, which are helpful for the clinician and the couple in their decision making about starting an ART treatment.Nos últimos anos, o hormonio anti-mülleriano (AMH) tem sido demonstrado como um marcador confiável da reserva ovariana. Neste estudo, avaliamos o risco de cancelamento do ciclo e as chances de boa resposta ovariana à hiperestimulação controlada e de gravidez, de acordo com o HAM no soro, medido antes de procedimentos de reprodução assistida em mulheres submetidas à injeção intracitoplasmática de espermatozóides. Um estudo analítico observacional incluiu mulheres submetidas a ICSI em um único centro. As análises dos subgrupos foram realizadas em pacientes agrupadas de acordo com os níveis de FSH ou para a sua idade. O risco de cancelamento do ciclo diminuiu de 64% em doentes com níveis séricos de HAM ≤% 3 pmol / L (0,42 ng / mL) a 21% com HAM ≥ 15 pmol / L (2,10 ng / mL). A taxa deboa resposta aumentou de quase nula em pacientes com HAM ≤ 3 pmol / L a 61% naqueles com AMH ≥ 15 pmol / L. A taxa de gravidez aumentou moderada, mas significativamente, de 31%, com HAM ≤ 3 pmol / L até 35% com HAM ≥ 15 pmol / L. Aqui fornecemos estimativas desses resultados de acordo com os valores de HAM no soro, em geral e em subgrupos de acordo com a idade do paciente ou de FSH, que são úteis para o clínico e para o casal em sua tomada de decisão sobre como iniciar um tratamento de RA.Fil: Calamera, Patricio. Centro Médico Seremas; ArgentinaFil: 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: de Vincentiis, Sabrina. Centro Médico Seremas; ArgentinaFil: Rey, Rodolfo Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas; ArgentinaFil: Brugo Olmedo, Santiago. Centro Médico Seremas; Argentin

Heads or tails? Structural events and molecular mechanisms that promote mammalian sperm acrosomal exocytosis and motility

Sperm structure has evolved to be very compact and compartmentalized to enable the motor (the flagellum) to transport the nuclear cargo (the head) to the egg. Furthermore, sperm do not exhibit progressive motility and are not capable of undergoing acrosomal exocytosis immediately following their release into the lumen of the seminiferous tubules, the site of spermatogenesis in the testis. These cells require maturation in the epididymis and female reproductive tract before they become competent for fertilization. Here we review aspects of the structural and molecular mechanisms that promote forward motility, hyperactivated motility, and acrosomal exocytosis. As a result, we favor a model articulated by others that the flagellum senses external signals and communicates with the head by second messengers to affect sperm functions such as acrosomal exocytosis. We hope this conceptual framework will serve to stimulate thinking and experimental investigations concerning the various steps of activating a sperm from a quiescent state to a gamete that is fully competent and committed to fertilization. The three themes of compartmentalization, competence, and commitment are key to an understanding of the molecular mechanisms of sperm activation. Comprehending these processes will have a considerable impact on the management of fertility problems, the development of contraceptive methods, and, potentially, elucidation of analogous processes in other cell systems.Fil: Buffone, Mariano Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina. University of Pennsylvania; Estados UnidosFil: Ijiri, Takashi W.. University of Pennsylvania; Estados UnidosFil: Cao, Wenlei. University of Pennsylvania; Estados UnidosFil: Merdiushev, Tanya. University of Pennsylvania; Estados UnidosFil: Aghajanian, Haig K.. University of Pennsylvania; Estados UnidosFil: Gerton, George L.. University of Pennsylvania; 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

Determination of a Robust Assay for Human Sperm Membrane Potential Analysis

Mammalian sperm must undergo a complex process called capacitation in order to fertilize the egg. During this process, hyperpolarization of the sperm plasma membrane has been mostly studied in mouse, and associated to its importance in the preparation to undergo the acrosome reaction (AR). However, despite the increasing evidence of membrane hyperpolarization in human sperm capacitation, no reliable techniques have been set up for its determination. In this report we describe human sperm membrane potential (Em) measurements by a fluorimetric population assay, establishing optimal conditions for Em determination. In addition, we have conducted parallel measurements of the same human sperm samples by flow cytometry and population fluorimetry, before and after capacitation, to conclusively address their reliability. This integrative analysis sets the basis for the study of Em in human sperm allowing future work aiming to understand its role in human sperm capacitation.Fil: Baró Graf, Carolina. 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; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Medicina Reproductiva; ArgentinaFil: Ritagliati, Carla. 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: Stival, Cintia Estefanía. 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: 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: 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: 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; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Medicina Reproductiva; Argentin

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

Determination of a Robust Assay for Human Sperm Membrane Potential Analysis

Mammalian sperm must undergo a complex process called capacitation in order to fertilize the egg. During this process, hyperpolarization of the sperm plasma membrane has been mostly studied in mouse, and associated to its importance in the preparation to undergo the acrosome reaction (AR). However, despite the increasing evidence of membrane hyperpolarization in human sperm capacitation, no reliable techniques have been set up for its determination. In this report we describe human sperm membrane potential (Em) measurements by a fluorimetric population assay, establishing optimal conditions for Em determination. In addition, we have conducted parallel measurements of the same human sperm samples by flow cytometry and population fluorimetry, before and after capacitation, to conclusively address their reliability. This integrative analysis sets the basis for the study of Em in human sperm allowing future work aiming to understand its role in human sperm capacitation.Fil: Baró Graf, Carolina. 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; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Medicina Reproductiva; ArgentinaFil: Ritagliati, Carla. 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: Stival, Cintia Estefanía. 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: 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: 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: 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; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Medicina Reproductiva; Argentin