Nitric oxide‑targeted protein phosphorylation during human sperm capacitation

Among many other molecules, nitric oxide insures the correct progress of sperm capacitation by mediating phosphorylation events. For a more comprehensive understanding of how this happens, we capacitated human spermatozoa from healthy men in the presence/absence of S-Nitrosoglutathione, a nitric oxide donor, two nitric oxide synthase inhibitors, NG- Nitro-l-arginine Methyl Ester Hydrochloride and Aminoguanidine Hemisulfate salt and, finally, with/without l-Arginine, the substrate for nitric oxide synthesis, and/or human follicular fluid. When analyzing the phosphorylation of protein kinase A substrates and tyrosine residues, we particularly observed how the inhibition of nitric oxide synthesis affects certain protein bands (~ 110, ~ 87, ~ 75 and ~ 62 kD) by lowering their phosphorylation degree, even when spermatozoa were incubated with l-Arginine and/or follicular fluid. Mass spectrometry analysis identified 29 proteins in these species, related to: spermatogenesis, binding to the zona pellucida, energy and metabolism, stress response, motility and structural organization, signaling and protein turnover. Significant changes in the phosphorylation degree of specific proteins could impair their biological activity and result in severe fertility-related phenotypes. These findings provide a deeper understanding of nitric oxide’s role in the capacitation process, and consequently, future studies in infertile patients should determine how nitric oxide mediates phosphorylation events in the species here described

Regulation of boar sperm functionality by the nitric oxidesynthase/nitric oxide system

Purpose Nitric oxide (NO) is a free radical synthesized mainly by nitric oxide synthases (NOSs). NO regulates many aspects in sperm physiology in different species. However, in vitro studies investigating NOS distribution, and how NO influences sperm capacitation and fertilization (IVF) in porcine, have been lacking. Therefore, our study aimed to clarify these aspects. Methods Two main experiments were conducted: (i) boar spermatozoa were capacitated in the presence/absence of S-nitrosoglutathione (GSNO), a NO donor, and two NOS inhibitors, N-G-nitro-L-arginine methyl ester hydrochloride (L-NAME) and aminoguanidine hemisulfate salt (AG), and (ii) IVF was performed in the presence or not of these supplements, but neither the oocytes nor the sperm were previously incubated in the supplemented media. Results Our results suggest that NOS distribution could be connected to pathways which lead to capacitation. Treatments showed significant differences after 30 min of incubation, compared to time zero in almost all motility parameters (P < 0.05). When NOSs were inhibited, three protein kinase A (PKA) substrates (similar to 75, similar to 55, and similar to 50 kDa) showed lower phosphorylation levels between treatments (P < 0.05). No differences were observed in total tyrosine phosphorylation levels evaluated by Western blotting nor in situ. The percentage of acrosome-reacted sperm and phosphatidylserine translocation was significantly lower with L-NAME. Both inhibitors reduced sperm intracellular calcium concentration and IVF parameters, but L-NAME impaired sperm ability to penetrate denuded oocytes. Conclusions These findings point out to the importance of both sperm and cumulus-oocyte-derived NO in the IVF outcome in porcine.This study was supported by H2020 MSC-ITN-EJD 675526 REP-BIOTECH, the Spanish Ministry of Economy and Competitiveness (MINECO), and the European Regional Development Fund (FEDER), Grant AGL2015-66341-R, and by a grant ESPDOC17/33 (to Jon Romero-Aguirregomezcorta) from the University of the Basque Country (UPV/EHU, Spain)

Papel del sistema de óxido nítrico en diferentes procesos reproductivos

El óxido nítrico (NO) regula los procesos fisiológicos y patológicos en diferentes sistemas orgánicos, incluido el sistema vascular, nervioso y reproductor. Su síntesis tiene lugar a partir del precursor L-Arginina, gracias a la familia de enzimas Óxido Nítrico Sintasa (NOS). Esta familia se encuentra formada por dos enzimas constitutivas, la NOS endotelial y la neuronal (eNOS, nNOS), y una inducible (iNOS). Los principales objetivos de esta tesis fueron estudiar el efecto del NO sobre la capacitación espermática en dos especies diferentes y determinar si los niveles de NO en el líquido folicular (FF) pueden predecir los resultados clínicos de las Técnicas de Reproducción Asistida. Para ello, en primer lugar, se analizó cómo la adición de NO o la inhibición de su síntesis afectaba a la capacitación espermática y a la fecundación in vitro (FIV) en la especie porcina (Capítulo 1). Posteriormente, se estudió cómo la fosforilación de las proteínas es modulada por el NO, L-Arginina y el FF durante la capacitación en espermatozoides humanos (Capítulo 2). Finalmente, se determinaron los niveles de NO en el FF y su relacion con la calidad de los ovocitos en mujeres (Capítulo 3). En el primer capítulo se describió la localización de las tres isoformas de la NOS en espermatozoides mediante inmunocitoquimica. Tanto la eNOS como la nNOS se ubicaron en la región de la cabeza espermática, con una señal débil en la pieza principal y final de la cola. Sin embargo, la iNOS mostró una distribución más general. En relación a: motilidad, sustratos de fosfo-PKA, fosforilación en tirosina, reacción acrosomal, translocación de fosfatidilserina y concentración del calcio intracelular, se observó que eran modificados por la presencia o ausencia de NO. Para estos análisis se utilizó el donante de NO, S-Nitrosoglutatión (GSNO), y dos inhibidores de las NOS, NG-Nitro-L-Arginina Metil Éster (L-NAME) y Aminoguanidina (AG). A tiempo 0 la motilidad no se vio afectada por los tratamientos utilizados. Sin embargo, a los 30 minutos se observó que la velocidad rectilínea (VSL) y la velocidad media (VAP) disminuyen en presencia de AG. La inhibición de NOS disminuyó el grado de fosforilación de tres sustratos de la PKA (~ 75, ~ 55 y ~ 50 kDa) aunque fosforilación de tirosina no difirió entre los tratamientos. El inhibidor L-NAME hizo disminuir el porcentaje de espermatozoides reaccionados y la exteriorización de la fosfatidilserina. L-NAME y AG indujeron una disminución en la concentración intracelular de calcio. En segundo lugar, se evaluó el papel del NO sobre la interacción de gametos en presencia o ausencia de células del cumulus. Se observó que tanto la adición del L-NAME como del AG durante la FIV, producía un descenso en el porcentaje de ovocitos penetrados cuando éstos se encontraban rodeados por las células del cumulus. No obstante, al eliminar éstas células la penetración disminuyó aún más, llegando a ser nula en presencia del inhibidor L-NAME. En el Capítulo 2, se analizó el papel de la L-Arginina y del FF sobre la fosforilación en serina, treonina y tirosina en espermatozoides de hombres y si su efecto se veía modificado por la presencia de donante de NO o por los inhibidores de síntesis de NO. Se observó que cuatro bandas de proteínas de ~ 110, ~ 87, ~ 75 y ~ 62 kDa disminuían la intensidad de fosforilación en presencia de inhibidores de las NOS, tanto en presencia como en ausencia de L-Arginina o FF. Posteriormente se identificaron que proteínas eran las afectadas y se observó que 29 de ellas estaban relacionadas con diferentes procesos reproductivos: espermatogénesis, unión a la zona pelúcida, metabolismo energético, respuesta al estrés, la motilidad y estructura, y señalización y recambio de proteínas. En el estudio final (Capítulo 3), se determinó la relación entre el nivel de nitrito (NO2) y nitrato (NO3) en el FF de mujeres que participaron en un programa de donación de gametos y la calidad de sus ovocitos. Se observó que ni el número total de ovocitos recogidos ni el de ovocitos MII se asociaron con los niveles de NO2 y NO3 en el FF. Sin embargo, la proporción de ovocitos MII sí que se relacionó directamente con los niveles de NO2 e inversamente con los niveles de NO3. Los resultados obtenidos en la presente tesis doctoral muestran la importancia del NO sobre los gametos y su interacción. En relación al espermatozoide porcino, se observó que la localización de las isoformas de la NOS es diferente y que la inhibición de la síntesis de NO disminuye los parámetros que participan en el proceso de capacitación y la penetración de ovocitos. En relación a los espermatozoides del hombre, se determinó que al inhibir la producción de NO se produce una disminución de la fosforilación de proteínas relacionadas con la función reproductiva y este efecto no se revierte por la presencia de L-Arginina o FF. Además, la síntesis de NO en el FF de mujeres donantes de ovocitos sometidas a tratamiento de superovulación, se relaciona con el porcentaje de ovocitos maduros, pero no con la cantidad total de ovocitos recuperados tras el tratamiento. Nitric oxide (NO) regulates both physiological and pathological processes in different organic systems, including the vascular, nervous, and reproductive system. Its synthesis takes place from a precursor, namely L-Arginine, thanks to the Nitric Oxide Synthase (NOS) family of enzymes. The latter includes two constitutive enzymes, the endothelial and the neuronal NOS (eNOS, nNOS), and one inducible isoform (iNOS). The main objectives of this thesis were to study the effect of NO on sperm capacitation in two different species and to determine whether or not the NO levels in the follicular fluid (FF) can predict the clinical outcomes from Assisted Reproduction Techniques. To do this, the first step was to analyze how the addition of NO or the inhibition of its synthesis affected sperm capacitation and in vitro fertilization (IVF) in the porcine species (Chapter 1). Subsequently, the modulation of protein phosphorylation by NO, L-Arginine and FF was investigated during human sperm capacitation (Chapter 2). Finally, the levels of NO in the FF were determined and their correlation with the quality of the oocytes in women was studied (Chapter 3). In the first chapter, the localization of the three NOS isoforms was described in spermatozoa by immunocytochemistry. Both eNOS and nNOS were located in the sperm head region, with a faint signal in the principal and end piece of the tail. On the other hand, iNOS showed a more general distribution. The following parameters: motility, phospho-PKA substrates, tyrosine phosphorylation, acrosome reaction, phosphatidylserine translocation and intracellular calcium concentration, were affected depending on the presence or absence of NO. For these assays, a NO donor, S-Nitrosoglutathione (GSNO), and two NOS inhibitors, NG-Nitro-L-Arginine Methyl Ester Hydrochloride (L-NAME) and Aminoguanidine Hemisulfate salt (AG), were used. At time 0, the used treatments did not affect motility patterns, however, after 30 minutes of incubation the straight-line (VSL) and average path velocity (VAP) decreased in the presence of AG. The inhibition of NOS lowered the phosphorylation degree of three PKA substrates (~ 75, ~ 55 and ~ 50 kDa), but tyrosine phosphorylation levels did not differ between the treatments. The inhibitor L-NAME decreased the percentage of acrosome-reacted sperm and phosphatidylserine translocation, whereas both L-NAME and AG reduced the sperm intracellular calcium concentration. Secondly, the role of NO on the interaction of gametes was evaluated in the presence or absence of cumulus cells. The addition of both L-NAME and AG during IVF induced a decrease in the percentage of penetrated oocytes, when the latter were surrounded by the cumulus cells. Nonetheless, when the cumulus cells were removed, the percentage of penetration was further diminished, becoming null in presence of the inhibitor L-NAME. In Chapter 2, the role of L-Arginine and FF was analyzed on the phosphorylation of serine, threonine and tyrosine residues in human spermatozoa and whether their effect was modified by the presence of a donor or inhibitors of NO synthesis. Four protein bands of ~ 110, ~ 87, ~ 75 and ~ 62 kDa showed a decrease in their phosphorylation degree when using NOS inhibitors, both in the presence or absence of L-Arginine and FF. Later, the affected proteins were identified, and it was observed that 29 of them were related to different reproductive processes: spermatogenesis, binding to the zona pellucida, energy and metabolism, stress response, motility and structural organization, signaling and protein turnover. In the final study (Chapter 3), the relationship between the FF levels of nitrite (NO2) and nitrate (NO3) in women participating in a gamete donation program and the quality of their oocytes was determined. Neither total nor MII oocyte yields were associated with the FF concentrations of NO2 and NO3. However, the proportion of MII oocytes was directly related to NO2 levels and inversely to NO3 levels. The results obtained in this doctoral thesis show the importance of NO on gametes and their interaction. In relation to porcine spermatozoa, it was observed that the localization of the NOS isoforms is different and that the inhibition of NO synthesis decreases the parameters involved in the capacitation process and the penetration of oocytes. As far as the human spermatozoa are concerned, the inhibition of NO synthesis leads to a decrease in the phosphorylation of proteins related to reproductive functions and, this effect is not reverted by the presence of L-Arginine or FF. In addition, the synthesis of NO in the FF of oocyte donors undergoing superovulation treatment is associated to the proportion of mature oocytes, but not to the total number of oocytes recovered after the treatmen

Chapter Nitric Oxide: Key Features in Spermatozoa

Several in vitro studies have pointed to the importance of nitric oxide (NO) in the female and male reproductive system in mammals. Its functions vary from preventing oocyte aging, improving the integrity of the microtubular spindle apparatus in aged oocytes, modulating the contraction of the oviduct, to regulating sperm physiology by affecting the motility, inducing chemotaxis in spermatozoa, regulating tyrosine phosphorylation, enhancing the sperm-zona pellucida binding ability, and modulating the acrosomal reaction. In spermatozoa, NO exerts its functions in different ways, which involve key elements such as the soluble isoform of guanylate cyclase, cyclic guanosine monophosphate (cGMP), cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), adenylate cyclase, and the extracellular signal-regulated kinase (ERK) pathway. Furthermore, NO leads to the S-nitrosylation of several sperm proteins, among them a substantial group associated with energy generation and cell movement, but also with signal transduction, suggesting a role for S-nitrosylation in sperm motility and in modulating the sperm function, respectively. In this chapter, an overview of how NO modulates the sperm physiology is presented, based on the knowledge acquired to this day