Role of the nitric oxide system in different reproductive processes

Abstract

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 treatment.